Compositions for inducing differentiation into retinal cells from retinal progenitor cells or inducing proliferation of retinal cells comprising Wnt signaling pathway activators

ABSTRACT

Disclosed is a composition for inducing the proliferation of retinal cells or the differentiation of retinal progenitor cells into retinal cells. The composition, similar to in vivo conditions for development during embryogenesis, induces stem cells to differentiate into a multitude of photoreceptor cells at high yield within a short period of time, without an additional gene transfer. In addition, the differentiated photoreceptor cells are useful in cellular therapy because they, when transplanted into degenerated or injured retinas, can be engrafted and fused within the retinas to prevent or cure retinal degeneration.

TECHNICAL FIELD

The present invention relates to compositions for inducing theproliferation of retinal cells and the differentiation of retinalprogenitor cells into retinal cells. More particularly, the presentinvention relates to compositions for inducing the differentiation ofretinal progenitor cells into retinal cells, especially photoreceptorcells and the proliferation of photoreceptor cells that comprise a Wntsignaling pathway activator.

BACKGROUND ART

Blindness is the medical condition of lacking visual perception forphysiological or neurological reasons. As many as tens of millions ofpeople, which accounts for 0.2-0.5% of the population of the world, areaffected with blindness, and are suffering from great losses inpersonal, social and economical respects. Retinal photoreceptordegeneration is one of the more dominant etiologies of blindness, causedinnately or by other various factors, including retinal dysplasia,retinal degeneration, aged macular degeneration, diabetic retinopathy,retinitis pigmentosa, congenital retinal dystrophy, Leber congenitalamaurosis, retinal detachment, glaucoma, optic neuropathy, and trauma.No drugs have been developed for the fundamental treatment of thesediseases thus far. To date, the replacement of dysfunctionalphotoreceptor cells, the alpha and omega of these retinal diseases, withnew ones is regarded as the only promising therapy. Photoreceptor cellimplantation is thought to prevent blindness or recover imperfecteyesight by delaying or restraining retinal degeneration, regeneratingdegenerated retina, and enhancing retinal functions.

Stem cells have become a candidate useful for cell therapy of retinaldiseases including bone marrow stem cells (BMSC), cord blood stem cells,amniotic fluid stem cells, fat stem cells, retinal stem cells (RSC),embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) andsomatic cell nuclear transfer cells (SCNT).

No significant research results have been yet suggested regarding thedifferentiation of stem cells into retinal cells (particularly,photoreceptor cells) and cell therapy based thereon. The differentiationof these stem cells into retinal cells might make it possible 1) toguarantee an infinite cell source for efficient cell therapy, 2) toidentify the differentiation mechanism from embryonic cells and retinalprogenitors into retinal cells, which has remained unclear, 3) to findretina differentiation-related genes and molecules and lesions thereby,4) to understand the pathogenesis of retinal degenerative diseases, and5) to develop drugs for preventing retinal degeneration and protectingthe retina.

Since the first establishment thereof, human embryonic stem cell lineshave been suggested to have the ability to differentiate into varioustypes of cells which are useful for the cellular therapy of variousdiseases. Human embryonic stem cells appear to have a high potentialwhen it comes to allowing the accurate examination of pathogeneticmechanisms and supplying fresh cells that can substitute fordysfunctional cells in clinical treatment. The production of human ESCderived-retinal photoreceptor cells under a completely identifiedreproducible condition and the use thereof in transplantation wouldguarantee a highly potential and effective therapy for retinalphotoreceptor cell-related diseases. It has been assumed that human ESCderived-cells will have the same properties and functions as did thecells formed that were formed through a normal differentiationprocesses. Based on this assumption, differentiation has been inducedunder circumstances similar to those of the developmental stages toproduce pancreatic hormone-expressing endocrine cells (D'Amour, et al.,Nat. Biotechnol., 2006; 24: 1392-401), neurons (Pankratz, et al., StemCells 2007; 25: 1511-20), muscle cells (Barberi et al., Nat. Med., 2007;13: 642-8), and vascular endothelial cells (Wang, et al., Nat.Biotechnol., 2007; 25: 317-8). Also, many attempts have been made todifferentiate human ESC into photoreceptor cells which may beeffectively used to treat retinal diseases, but this ended with failurefor most cases.

In fact, differentiation into retinal progenitor cells from humanembryonic stem cells is the greatest achievement made thus far in thisfield, but the differentiation of retinal progenitor cells intophotoreceptor cells failed (differentiation rate of less than 0.01%)(Lamba et al., Proc. Natl. Acad. Sci. USA, 2006; 103: 12769-74). Onereport held that human embryonic stem cells were successfully induced todifferentiate into photoreceptor cells, but the method used thereinrequires more than 200 days in total for the differentiation, with adifferentiation rate of as low as 8%, and thus is impossible to apply tothe clinical treatment of blindness (Osakada et al., Nat. Biotechnol.,2008; 26: 215-24).

The Wnt signaling pathway participates in regulating various processesduring embryogenesis, including tissue development, cell proliferation,morphology, motility and cell-fate determination, etc (Wodarz & Nusse,Annu. Rev. Cell Dev. Biol., 1998; 14: 59-88). It is known to promote orregulate differentiation depending on tissue type and differentiationlevel. To date, in the context of embryonic development and cellbiology, the Wnt signaling pathway including Wnt3a has been reported tobe deeply involved in the regulation of cellular differentiation and themaintenance and proliferation of undifferentiated cells (Aubert, et al.,Nat. Biotechnol., 2002; 20: 1240-5). Nowhere has the effect of the Wntsignaling pathway on cell differentiation and maturation associated withvertebrate eye patterning and neurogenesis been reported in the priorart.

DISCLOSURE Technical Problem

Leading to the present invention, intensive and thorough research intothe differentiation of human ESC into retinal cells, particularlyphotoreceptor cells, conducted by the present inventors, usingchemically defined, resulted in the finding that when in vitroconditions for differentiation into photoreceptor cells, similar to invivo conditions, in which differentiation-associated factors and theirinhibitors were employed, was applied, the Wnt signaling pathwayactivators played an important role in the proliferation of retinalcells and the differentiation and maturation from stem cells intoretinal cells, particularly photoreceptor cells.

Technical Solution

It is therefore an object of the present invention to provide acomposition for inducing the proliferation of retinal cells, comprisinga Wnt signaling pathway activator.

It is another object of the present invention to provide a compositionfor inducing differentiation from retinal progenitor cells into retinalcells, particularly photoreceptor cells, comprising a Wnt signalingpathway activator.

Advantageous Effects

As described above, the composition of the present invention allows stemcells to differentiate into a multitude of photoreceptor cells at highyield within a short period of time, without an additional genetransfer. Able to generating a multitude of photoreceptor cells at highyield, the composition of the present invention does neither require theisolation of photoreceptor cells through flow cytometry nor anadditional photoreceptor proliferation so that the differentiatedphotoreceptor cells may be readily transplanted into degenerated orinjured retinas. In addition, the differentiated photoreceptor cells areuseful in cellular therapy because they, when transplanted intodegenerated or injured retinas, can be engrafted and fused within theretinas to prevent or cure retinal degeneration.

DESCRIPTION OF DRAWINGS

FIG. 1 is of cytomorphological microphotographs:

(A) Left. Typical cell floc of hESCs in an undifferentiated state (29passages; 40× magnification), after being cultured for 5 days from cellsof passage number 28. Characterized by a definite separation fromadjacent MEF feeder cells. Having plain surface and uniform morphology.

(A) Right. Floating aggregates (40× magnification), being cultured for 4days in ultra-low attachment plates after isolation from the hESC flocof FIG. 1A Left. Spherical morphology. Consisting of approx. 292 53cells per floating aggregate.

(B)-(D). Morphological microphotographs of cells differentiating intoretinal cells.

(B). Cells on Day 14 after induction of the differentiation, that is,after the floating aggregates were transferred topoly-D-lysine/laminin-coated plates and cultured for 10 days therein,which was on Day 14 after the induction of the differentiation of theundifferentiated hESCs. The cells were observed to be separated from thefloating aggregates and to undergo differentiation. Morphology of cellsin the early stage of differentiation, with meager cytoplasm and round,large nuclei.

(C). Cells on Day 19 after the induction of the differentiation, thatis, cells after the undifferentiated hESCs were induced to differentiatefor 19 days. They differentiated into retinal progenitor cells, withconcomitant active proliferation. Cell flocs under active proliferationand differentiation formed an eddy formation or a rosette configuration.

(D). Cells on Day 21 after induction of the differentiation, showing anincreased number of cells resulting from active proliferation. The cellsbecame richer in cytoplasm and the size of their nucleus was smallerthan those of FIG. 1C as the differentiation progressed. The cellsappeared to function in response to light.

(E)-(H). Various morphologies of cell flocs on Day 29 after induction ofthe differentiation.

(E). The morphology of most cells, particularly observed in denselypopulated regions. With the progress of differentiation, the cellsshowed the same cellularity, but had a richer cytoplasm and smaller anddenser nuclei, compared to those on Day 21 after induction.

(F). The morphology of cells in a scarcely populated region. The cellflocs showed directivity and moved towards a certain point whichdepended on the cell cluster. More plentiful, opposite end-pointedcytoplasms and spindle-like nuclei were observed.

(G). Cell flocs, some having a multiple of nerve bundles.

(H) Left. Cell flocs some of which show long neural axons.

(H) Right. Cell flocs some of which show the morphology ofdifferentiated neurons.

-   -   Microscopic field: (A) (left, right: 40× magnification); (B)-(G)        (left: 50× magnification; right: 200× magnification); (H) (left        and right: 200× magnification).

FIG. 2 is a graph showing changes in expression levels of the retinalcell markers Crx, recoverin, rhodopsin, peripherin2 and Ki67 withculture time period.

FIG. 3 is a set of microphotographs showing the cells obtained bydifferentiation into retinal cells for 29 days, which were immunostainedfor recoverin and rhodopsin, both indicative of photoreceptor cells.

After hESCs were induced to differentiate into photoreceptor cells, anexamination was made of the expression of photoreceptor cell-specificproteins. More than 80% of the differentiated cells tested positive toboth recoverin (a universal photoreceptor cell marker) and rhodopsin(characteristic of rod photoreceptor cells).

(A) and (B). Flocs of differentiated photoreceptor cells.

(C). Individual cells in scarcely populated regions.

Recoverin and rhodopsin are distinctively expressed in thedifferentiated photoreceptor cells.

-   -   Microscopic field: (A) 100× magnification; (B) 200×        magnification; (C) 400× magnification.    -   Merge: superimposed photographs of cells which were fluorescent        immunostained for recoverin and rhodopsin, cells expressing both        the antigens being represented yellow (green+red).    -   ⁺Merge/DAPI: DAPI is a nucleus-stained cell population. The        Merge/DAPI images are of superimposed fluorescence photographs        to detect the expression of both recoverin and rhodopsin and the        expression of DAPI, showing cell contours and the expression        pattern of the two antigens, simultaneously.

FIG. 4 is of fluorescence microphotographs of the cells obtained afterinducing differentiation into retinal cells for 29 days, showing theexpression of the photoreceptor cell markers rhodopsin, rom-1 andperipherin2.

The differentiated photoreceptor cells were observed to express Rom-1and peripherin2, both characteristic of the outer segment ofrhodopsin-positive rod photoreceptor cells.

(A). Cell flocs positive to both rhodopsin and rom-1.

(B). Individual cells positive to both rhodopsin and rom-1. Within eachcell, rhodopsin and rom-1 were expressed at distinctly differentpositions. With the progress of differentiation, rhodopsin was expressedin the inner cytoplasm while rom-1 was positioned at the outermostcytoplasm.

(C). Flocs of the differentiated photoreceptor cells which were positiveto both rhodopsin and peripherin2.

(D). Individual cells positive to both rhodopsin and peripherin2.

-   -   Microscopic field: (A) 100× magnification; (B) 400×        magnification; (C) 100× magnification; (D) 400× magnification.

FIG. 5 is of fluorescence microphotographs of the cells obtained afterinducing differentiation into retinal cells for 29 days, showing theexpression of the photoreceptor cell markers rhodopsin, phosducin andPde6b. These proteins are responsible for the response to light,demonstrating that the differentiated photoreceptor cells are exhibitingtheir proper functions.

(A). Flocs of the differentiated photoreceptor cells which are positiveto both rhodopsin and phosducin.

(B). Individual cells positive to both rhodopsin and phosducin.

(C). Flocs of the differentiated photoreceptor cells positive to bothrhodopsin and Pde6b.

(D). Individual cells positive to rhodopsin and Pde6b.

-   -   Microscopic field: (A) 100× magnification; (B) 400×        magnification; (C) 100× magnification; (D) 400× magnification.

FIG. 6 is of fluorescence microphotographs of cells obtained afterinducing differentiation into retinal cells for 29 days, showing theexpression of the photoreceptor cell markers rhodopsin andsynaptophysin.

(A). Flocs of the differentiated photoreceptor cells positive to bothrhodopsin and synaptophysin. The expression of these proteinsdemonstrates that the differentiated photoreceptor cells are in synapseinteraction with other retinal neurons and are participating in theformation of retinal nerve circuits.

(B). Individual cells positive to rhodopsin and synaptophysin.

-   -   Microscopic fields: (A) 100× magnification; (B) 400×        magnification.

FIG. 7 is of fluorescence microphotographs of cells which were obtainedafter inducing differentiation into retinal cells for 29 days andimmunostained against cone photoreceptor cells.

Left panel. Cell flocs positive to blue opsin.

Right panel. Individual cells positive to blue opsin.

The expression of blue-opsin is evidence that the differentiated cellsare blue opsin-cone photoreceptor cells.

-   -   Microscopic field (left) 100× magnification; (right) 400×        magnification.

FIG. 8 is of fluorescence microphotographs of cells which were obtainedafter inducing differentiation into retinal cells for 29 days and whichwere immunostained against characteristic photoreceptor cells. Varioustypes of cells which had undergone further differentiation wereobserved.

Left. Cells positive to both recoverin and rhodopsin, showing amorphology characteristic of photoreceptor cells.

Middle. Cells positive to synaptophysin and rhodopsin, showing furthermature differentiation.

Right. Rhodopsin-positive cells characterized by rich rhodopsinmolecules within the cytoplasm.

-   -   Microscopic field 400× magnification.

FIG. 9 is of fluorescence microphotographs of cells which were obtainedafter inducing differentiation into retinal cells for 29 days and whichwere immunostained against neural retinal progenitor cells andphotoreceptor cell precursors.

(A). Cell flocs positive to both Rax and Pax6.

(B). Individual cells positive to both Rax and Pax6.

Most cells were observed to express both the antigens although theexpression level was different between them, indicating that the retinalcells obtained after differentiation induction for 29 days were derivedfrom neural retinal progenitor cells.

(C). Cell flocs positive to the proliferative cell marker Ki67 and thephotoreceptor cell precursor marker Crx.

(D). Individual cells positive to both Ki67 and Crx.

Most Crx-positive cells do not express Ki67. This coincides with thefact that photoreceptor cell precursors express Crx immediately afterleaving the cell proliferation cycle. Sometimes, a minority ofCrx-positive cells still continue to express Ki67.

-   -   Microscopic field (A) 100× magnification; (B) 400×        magnification; (C) 100× magnification; (D) 400× magnification.

FIG. 10 is of fluorescence microphotographs of cells which were obtainedafter inducing differentiation into retinal cells for 29 days andimmunostained against retinal cells other than photoreceptor cells.

(A). Cell flocs (left) and individual cells, positive to both Islet-1and NF-200, which gives evidence of retinal ganglion cells becausenuclei and axons are positive to Islet-1 and NF-200, respectively.

(B). Cell flocs (left) and individual cells (right) positive to PKC-α,which gives evidence of bipolar cells.

(C). Cell flocs (left) and individual cells (right) positive to Prox-1,which gives evidence of horizontal cells.

(D). Cell flocs (left) and individual cells (right) positive to GFAP,which gives evidence of Muller glial cells.

(E). Cell flocs (left) and individual cells (right) positive to bothRpe65 and ZO-1, which gives evidence of retinal pigmented epithelium.

-   -   Microscopic field: (A). Left. 100× magnification, Right. 400×        magnification; (B) Left. 100× magnification, Right. 400×        magnification; (C) Left. 100× magnification, Right. 400×        magnification; (D) Left. 100× magnification, Right. 400×        magnification; (E) Left. 100× magnification, Right. 400×        magnification.

FIG. 11 is of fluorescence microphotographs of the cells resulting frominducing differentiation into retinal cells for 29 days, with BIO andpurmorphamine used respectively instead of Wnt3a and Shh, which wereimmunostained against photoreceptor cell precursors and photoreceptorcells.

(A). Cell flocs positive to both the proliferative cell marker Ki67 andthe photoreceptor cell precursor-specific antigen Crx.

(B). Individual cells positive to both Ki67 and Crx.

(C). Cell flocs positive to the photoreceptor cell markers recoverin andrhodopsin.

(D). Individual cells positive to recoverin and rhodopsin.

-   -   Microscopic field (A) 100× magnification; (B) 400×        magnification; (C) 100× magnification; (D) 400× magnification.

FIG. 12 is of fluorescence microphotographs of the cells resulting frominducing differentiation into retinal cells for 29 days, with BIO andpurmorphamine used respectively instead of Want3a and Shh, which wereimmunostained for the photoreceptor cell markers rhodopsin, peripherin2and rom-1.

(A). Cell flocs positive to both rhodopsin and peripherin2.

(B). Individual cells positive to both rhodopsin and peripherin2.

(C). Cell flocs positive to both rhodopsin and rom-1.

(D). Individual cells positive to both rhodopsin and rom-1.

-   -   Microscopic field: (A) 100× magnification; (B) 400×        magnification; (C) 100× magnification; (D) 400× magnification.

FIG. 13 is of fluorescence microphotographs of the cells resulting frominducing differentiation into retinal cells for 29 days, with BIO andpurmorphamine used respectively instead of Want3a and Shh, which wereimmunostained against cone photoreceptor cells.

Left. Blue-opsin-positive cell flocs.

Right. Blue-opsin-positive individual cells.

-   -   Microscopic field (Left) 100× magnification; (Right) 400×        magnification.

FIG. 14 is of microphotographs of human iPSCs.

(A). Cytomorphological microphotographs. (A) Left. Typical cell floc ofhuman iPSCs in an undifferentiated state (passages 43; Microscopicfield: 40 magnifications), after being cultured for 6 days from cells ofpassages 43. Characterized by definite separation from adjacent MEFfeeder cells. Having a plain surface and uniform morphology, which isalso characteristic of undifferentiated hESCs. (A) Right. Floatingaggregates (Microscopic field: 40× magnification), being cultured for 4days in ultra-low attachment plates after isolation from the human iPSCfloc of FIG. 14A Left.

(B). Fluorescence microphotographs of undifferentiated human iPSCsimmunostained for characteristic markers. Cell flocs in which most cellsare positive to both SSEA4 and Nanog, which gives evidence of thecontinuance of undifferentiated states.

-   -   Microscopic field (leftmost) 40× magnification; (the others)        100× magnification.

FIG. 15 is a set of fluorescence microphotographs showing the cellsobtained by inducing human iPSC to differentiate into retinal cells for29 days, which were immunostained for recoverin and rhodopsin, bothcharacteristic of photoreceptor cells.

The photoreceptor cells differentiated from human iPSCs were assayed forthe expression of photoreceptor cell-specific proteins.

(A). Flocs of differentiated photoreceptor cells.

(B). Individual cells at the low cell density area.

(C). Individual cells in scarcely populated regions.

Recoverin and rhodopsin are distinctively expressed in thedifferentiated photoreceptor cells.

-   -   Microscopic field: (A) 100× magnification; (B) 400×        magnification; (C) 400× magnification.

FIG. 16 is of photographs showing RT-PCR for genes specific for retinalcells. The cells generated by inducing undifferentiated hESCs todifferentiate into retinal cells for 29 days were assayed for the mRNAexpression levels of genes associated with retinal progenitor cells,photoreceptor cells and other retinal cells using RT-PCR.

(A). RT-PCR products of the retinal progenitor cell-specific genes RAX(495 bp), PAX6 (275 bp), SIX3 (307 bp), SIX6 (272 bp), LHX2 (285 bp) andCHX10 (281 bp). Inter alia, RAX and PAX6 were expressed to the same highextent as was the quantitative control gene GAPDH (PCR product size: 302bp). In contrast, none of the developing cerebral cortex-relevant geneARX (462 bp), the developing mesodermal gene T (541 bp), or thedeveloping endodermal gene AFP (318 bp) were found in the RT-PCRproducts, suggesting that the method of the present invention isspecifically directed to the mRNA expression of retina-relevant genes.

(B). RT-PCR products of genes associated with photoreceptor cells andother retinal cells. The photoreceptor cell-relevant genes CRX (353 bp),NRL (206 bp), RCVRN (150 bp), RHO (258 bp), PDE6B (409 bp), SAG (400 bp)and OPN1SW (206 bp) were observed to be amplified by RT-PCR. The retinalganglion cell genes ATHO7 (246 bp) and POU4F2 (175 bp), the amacrinecell gene NEUROD1 (523 bp), and the bipolar cell gene ASCL1 (467 bp)were also found in the RT-PCR products.

Characteristics of photoreceptor cell-relevant genes are as follows: CRXand NRL are transcription genes characteristic of photoreceptor cellprecursors and rod photoreceptor cells, respectively. RCVRN (recoverin)is a universal photoreceptor cell gene that tests positive for both coneand rod photoreceptor cells. RHO (rhodopsin) is rod photoreceptor cellspecific. PDE6B and SAG (human arrestin) are involved in thephototransduction of photoreceptor cells. The expression of these genesgives evidence of the development and maturation of the photoreceptorcell's own functions. OPN1SW is characteristic of short wave (blueopsin)-cone photoreceptor cells. M: marker.

FIG. 17 shows RT-PCR and base sequencing results of genes characteristicof photoreceptor cells.

RT-PCR was performed on the cells generated by inducing undifferentiatedhESCs to differentiate into retinal cells for 29 days, so that thephotoreceptor cell-specific genes RCVRN (NM_(—)002903.2) and RHO(NM_(—)000539.3) could be detected. The RT-PCR products were identifiedas RCVRN and RHO by base sequencing.

(A). Agarose gel electrophoresis of the RT-PCR products showing RCVRN at150 bp and RHO at 258 bp. M: marker.

(B). Chromatogram of base sequencing analysis, showing an RCVRN basesequence (top) and an RHO base sequence (bottom). The RCVRN and RHO genebase sequences were found to perfectly coincide with human standardsequences (http://www.ncbi.nlm.nih.gov/), indicating that thephotoreceptor cells express human RCVRN and RHO genes.

FIG. 18 is of electroretinograms of the retinal degeneration mouserd/SCID which had been or had not been transplanted with thehESC-derived photoreceptor cells.

(A). Electroretinograms of 8-week-old, non-transplanted mice. Nocharacteristic ERG wave forms were found. The ERG b-wave had anamplitude of 6.29 μV for the right eye and 0.0542 μV for the left eye.

(B). Electroretinograms of 8-week-old mice 4 weeks after thetransplantation. Compared to the non-transplanted right eye, the EGRb-wave from the photoreceptor cell-transplanted left eye formedcharacteristic wave forms, with an amplitude of as high as 74.5 μV. Therd/SCID mice transplanted with the hESC-derived photoreceptor cellsshowed definite responses to light stimuli as measured byelectroretinography.

FIG. 19 is a graph comparing the amplitude of the b-wave between rd/SCIDmice with retinal degeneration which had been transplanted or had notbeen transplanted with the hESC-derived photoreceptor cells.

The ERG b-wave from the photoreceptor cell-transplanted rd/SCID miceformed characteristic wave forms, with an amplitude of 48.4(±3.4) μV(sample size=13). In contrast, characteristic wave forms where nowhereto be found in the ERG of the non-transplanted group, which showed ab-wave amplitude of 10.3(±2.5) μV (sample size=17) which is differentfrom that of the transplanted group with statistical significance(p<0.0001) (Table 6, FIG. 19).

FIG. 20 is of fluorescence microphotographs after the hESC-derivedphotoreceptor cells had been transplanted into the mouse model ofretinal degeneration (rd/SCID).

Four weeks after the transplantation, the hESC-derived photoreceptorcells were analyzed for engraftment into the retina using rhodopsin andrecoverin, both characteristic of human mitochondria and photoreceptorcells. When cells positive to rhodopsin and recoverin showed a positiveresponse to a human mitochondrial antigen, they were decided to behESC-derived photoreceptor cells.

(A). Immunostained human-specific mitochondria and rhodopsin in thetransplanted group. A new outer nuclear layer (ONL) was formed of a 4-or 5-fold, rhodopsin-positive photoreceptor cell layer.

(B). Immunostained human-specific mitochondria and rhodopsin in thenon-transplanted group of rd/SCID mice of the same age (8 weeks old)which served as a control. Only a single outer nuclear layer wasobserved, consisting mostly of cone photoreceptor cells. Almost no rodphotoreceptor cells were observed due to degeneration while only tworesidual cells which were undergoing degeneration were detected.

(C). Immunostained human-specific mitochondria and recoverin in thetransplanted group. A 4- or 5-fold recoverin-positive cell layer formeda new outer nuclear layer. In the transplanted group, a 4- or 5-foldrecoverin-positive cell layer was formed in the outer nuclear layer aswell as in the inner nuclear layer (INL).

(D). Immunostained human-specific mitochondria and recoverin in thenon-transplanted group used as a control. Positive responses weredetected in a total of 40 cells. A single recoverin-positive outernuclear layer consisted of cone-photoreceptor cells while therecoverin-positive inner nuclear layer was formed of cone-bipolar cells.

-   -   Microscopic field: (A) and (C) Left. 200× magnification;        (A)-(D): 400× magnification.    -   ONL: outer nuclear layer

INL: inner nuclear layer

RGC: retinal ganglion cell

FIG. 21 is a graph showing engraftment results after human ESC-derivedphotoreceptor cells were transplanted into the mouse model of retinaldegeneration (rd/SCID).

In the non-transplanted group, rhodopsin was detected in only two of atotal of 199 cells per observation microscopic field (positive rate:1.0%). On the other hand, 88 of a total of 215 cells per microscopicfield were rhodopsin positive in the transplanted group (positive rate:40.8%) (p<0.0001). Accordingly, the transplanted rod photoreceptor cellswere found to occupy approximately 40% of the total area of the retinalsections. Positive responses to recoverin were detected in 40 of thetotal of 168 cells per microscopic field in the non-transplanted group(positive rate: 23.8%), but in 120 of the total 292 cells permicroscopic field in the transplanted group (positive rate: 41.0%), withstatistical significance (p<0.0001).

FIG. 22 is of fluorescence microphotographs after the hESC-derivedphotoreceptor cells had been transplanted into the mouse model ofretinal degeneration (rd/SCID).

Four weeks after the transplantation, human mitochondria and thephotoreceptor cell antigen synaptophysin were immunostained andanalyzed. In the non-transplanted group, recoverin means bipolar cellsin the inner nuclear layer and cone photoreceptor cells in the outernuclear layer. In the transplanted group, a 4- or 5-foldsynaptophysin-positive cell layer was found to form a new outer nuclearlayer, suggesting that the photoreceptor cells in the newly formed outernuclear layer are in synaptic interaction with other intraretinal cellswithin the retinas of the transplanted mice.

-   -   Microscopic field: Left. 200× magnification; the others 400×        magnification.

FIG. 23 is a graph showing mean cell counts according to combinations ofthe differentiation factors Wnt3a (W), Shh (S) and retinoic acid (R).

FIG. 24 is a graph showing positive rates of photoreceptor cell markersversus the concentrations of Wnt3a and its inhibitor Dkk-1, in theabsence of Shh and retinoic acid (*: P<0.0001; **: P=0.0381)

BEST MODE

In accordance with an aspect thereof, the present invention pertains toa composition for promoting the proliferation of retinal cells,comprising a Wnt signaling pathway activator.

The term “Wnt signaling pathway activator”, as used herein, is intendedto refer to a substance activating the Wnt signaling pathway which hasbeen found to regulate various processes during embryogenesis, includingcell-fate determination, reconstruction of organization, polarity,morphology, adhesion and growth, and the maintenance and proliferationof undifferentiated cells (Logan & Nusse, Annu Rev Cell Dev Biol. 2004;20: 781-810). As long as it transduces Wnt-mediated orbeta-catenin-mediated signals, any activator may be included within theWnt signaling pathway. The Wnt signaling pathway is a series ofprocesses that are initiated by the binding of the trigger Wnt to itsreceptor or mediated by the stabilization of the downstream factorβ-catenin. The following is a description of how to activate Wntsignaling pathway.

1) By adding a Wnt protein: Wnt, a first trigger of the Wnt signalingpathway, is a family of secreted glycoproteins. 19 Wnts have beenidentified: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6,Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, andWnt16b.

2) By increasing the level of β-catenin: most cells respond to Wntsignaling pathway by an increase in the level of β-catenin. That is, anincrease in dephosphorylated β-catenin level or the stabilization ofβ-catenin means the translocation of β-catenin into the nucleus.

3) By phosphorylation of dishevelled or phosphorylation of aWnt-accosiated receptor, LRP tail.

4) By using GSK3 (glycogen synthase kinase 3) inhibitors: lithium (Li),LiCl, bivalent Zn, BIO (6-bromoindirubin-3′-oxime), SB216763, SB415286,QS11 hydrate, TWS119, Kenpaullone, alsterpaullone, indirubin-3′-oxime,TDZD-8, and Ro 31-8220 methanesulfonate salt.

5) By blocking negative regulators of Wnt signaling pathway, such asAxin and APC, or by using RNAi.

6) With activators of the Wnt pathway, such as norrin and R-spondin2:Norrin binds to Frizzled4 receptor while R-spondin2 interacts withFrizzled8 and LRP6.

7) By gene transfer, including transfection: one can activate Wntsignaling pathway using either Wnt overexpression constructs orβ-catenin overexpression constructs.

In a preferred embodiment, the Wnt signaling pathway activators may beemployed without limitation. Preferred are Wnt1, Wnt2, Wnt2b, Wnt3,Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a,Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16b; substances increasing β-cateninlevels; GSK3 inhibitors such as lithium, LiCl, bivalent zinc, BIO,SB216763, SB415286, CHIR99021, QS11 hydrate, TWS119, Kenpaullone,alsterpaullone, indirubin-3′-oxime, TDZD-8 and Ro 31-8220methanesulfonate salt; Axin inibiotors, APC inhibitors, norrin andR-spondin 2, and the most preferred are Wnt3a, Wnt1, Wnt5a, Wnt11,norrin, LiCl, BIO and SB415286.

In a preferred embodiment of the present invention, the composition forinducing the proliferation of retinal cells contains the Wnt signalingpathway activator except for LiCl, BIO and SB415286 in an amount of from0.01 to 500 ng/ml, preferably in an amount of from 0.1 to 200 ng/ml, andmore preferably in an amount of from 1 to 100 ng/ml. Among the Wntsignaling pathway activators, LiCl is used in the medium in an amount of0.1 to 50 mM, preferably in an amount of 0.5 to 10 mM, and morepreferably in an amount of 1 to 10 mM; BIO is used in an amount of 0.1to 50 μM, preferably in an amount of 0.1 to 10 μM, and more preferablyin an amount of 0.5 to 5 μM; SB415286 is used in an amount of 0.1 to 500μM, preferably in an amount of 1 to 100 μM, and more preferably in anamount of 5 to 50 μM. In a modification of the embodiment, the mediummay contain 50 ng/ml of Wnt3a or Wnt1; 50 or 100 ng/ml of Wnt5a andWnt11; 50 ng/ml of norrin; 2.5 or 5 mM of LiCl; 2 μM of BIO, or 30 μM ofSB415286. The term “retina”, as used herein, refers to light-sensitivetissue. The retina is the innermost (sensory) transparent coat in theeyeball and is directly relevant to vision. Just outside theneurosensory retina is the retinal pigment epithelium consisting ofpigmented cells. In a broader sense, the retina includes the innersensory coat and the outer retinal pigmented epithelium. The retina islocated at the back of the eye and originates as outgrowths of thedeveloping brain in embryonic development. The retina is like afive-layered cake, consisting of three nuclear layers with two networklayers intercalated therebetween. The three nuclear layers are: theoutermost nuclear layer consisting of photoreceptor cells; inner nuclearlayer consisting of horizontal cells, bipolar cells, amacrine cells, andMuller glias; and the innermost retinal ganglion layer consisting of thenuclei of retinal ganglion cells. After passing through the cornea andthe lens of the eye, light reaches the outer nuclear layer through theretinal ganglion layer and the inner layer in that order, producingneural impulses at photoreceptor cells. These neural impulses aretransduced in a reverse direction. That is, when photoreceptor cells arestimulated by the neural impulses, nerve currents are transmitted to theinner nuclear layer and then into optic nerve fibers through the retinalganglion cell layer.

As used herein, the term “retinal cell” is intended to refer to a cellconstituting a part of the retina, including photoreceptor cells such asrod and cone photoreceptor cells, retinal ganglion cell, horizontalcells, bipolar cells, amacrine cells, Muller glial cells, and retinalpigmented epithelium. The composition of the present invention inducesretinal cells, particularly photoreceptor cells to proliferate.

As used herein, the term “photoreceptor cell” refers to a specializedtype of neuron found in the eye's retina that is capable ofphototransduction and allowing shapes and colors to be recognized: whenlight reaches the retina through the cornea and the lens, thephotoreceptor cell converts the light energy into electric energy whichis then transmitted into the brain. There are two main types ofphotoreceptor cells: rods and cones, which are adapted for low light andbright light, respectively. Cone cells gradually become denser towardsthe center of the retina, that is, the yellow spot, and function toperceive images and colors while rod cells are distributed predominantlyat the periphery of the retina, allowing the perception of images andlight. Photoreceptor cells are characterized by the ability to expressat least one, two or three markers selected from among recoverin (rodphotoreceptor cells, cone photoreceptor cells), rhodopsin (rodphotoreceptor cells), peripherin2 (rod photoreceptor cells), rom1 (rodphotoreceptor cells, cone photoreceptor cells), Pde6b (rod photoreceptorcells), arrestin sag (rod photoreceptor cells), phosducin (rodphotoreceptor cells, cone photoreceptor cells), synaptophysin (rodphotoreceptor cells, cone photoreceptor cells), red/green opsin (conephotoreceptor cells), and blue opsin (cone photoreceptor cells).

In a preferred embodiment, the retinal cells or photoreceptor cells ofthe present invention may be derived from animals including humans. Asused herein, the term “animal” is intended to include humans, primates,cows, pigs, sheep, horses, dogs, mice, rats, and cats, humans beingpreferred.

In an embodiment of the present invention, a Wnt signaling pathwayactivator was identified as playing an important role in theproliferation of retinal cells. In this regard, the Wnt signalingpathway activator Wnt3a (W), the Shh signaling pathway activator Shh(S), and retinoic acid (R) were combined with one another to affordvarious compositions which were then applied to hESCs at different timepoints, followed by culturing the cells for 29 days. After completion ofthe differentiation, differentiated retinal cells were counted (Example9).

The highest cell population was measured upon the use of W+/S+/R+(3.98(±0.64)×10⁶ cells), followed by 2.74(±0.36)×10⁶ cells in W+/S−/R+and 2.21(±0.67)×10⁶ cells in W+/S+/R−. W−/S−/R− and W−/S+/R+proliferated the cells at a density of as low as 0.87(±0.38)×10⁶ and0.73(±0.16)×10⁶, respectively (FIG. 23 and Table 8). The data indicatethat cell proliferation is independent of the presence of S and R, butdepends on W. A great part of the cell proliferation results from theeffect of Wnt3a. When comparing the two media W+/S+/R+ and W−/S+/R+, thecell populations (3.98×10⁶ vs. 0.73×10⁶) differ by five times (FIG. 23and Table 8). This is another evidence that Wnt3a plays a critical rolein cell proliferation. After being cultured four weeks in the (W+/S+/R+)culture for 4 weeks, the undifferentiated cells proliferated to 3.98×10⁶differentiated cells (FIG. 23 and Table 8), which was 257-fold higherthan the population of the initial cells, for the first time indicatingthat the Wnt signaling pathway activator can be used for proliferatingretinal cells.

As used herein, the term “stem cell” refers to a cell with pluripotencyto give rise to all derivatives of the three primary germ layers (theendoderm, mesoderm and ectoderm) or one with multipotency todifferentiate into mature cells closely related in tissue type andfunction.

As used herein, the term “animal” is intended to include humans,primates, cows, pigs, sheep, horses, dogs, mice, rats, and cats, humansbeing preferred.

As used herein, the term “embryonic stem cell” refers to a pluripotentcell derived from the inner cell mass of the blastocyst immediatelybefore the nidation of a fertilized egg on the uterine wall, which candifferentiate into any type of animal cells, and is intended in abroader sense to include stem cell-like cells such as embryoid bodiesand induced pluripotent stem (iPS) cells.

The term “adult stem cell”, as used herein, is intended to refer to amultipotent cell which is isolated from tissues and cultured ex vivo,and to include bone marrow stem cells, cord blood stem cells, amnioticfluid stem cells, fat stem cells, retinal stem cells, intraretinalMuller glial cells and neural stem cells.

In accordance with another aspect, the present invention pertains to acomposition for inducing retinal progenitor cells to differentiate intoretinal cells, comprising a Wnt singling pathway activator. Preferably,the retinal cells are photoreceptor cells.

The term “retinal progenitor cell”, as used herein, is intended to referto a multipotent progenitor cell which can differentiate into cellspresent in the retina and retinal pigmented epithelial cells. Retinalprogenitor cells may be cells which have been differentiated fromvarious stem cells, that is, retinal stem cells, cord blood stem cells,amniotic fluid stem cells, fat stem cells, bone marrow stem cells,adipose stem cells, neural stem cells, embryonic stem cells, inducedpluripotent stem cells, or somatic cells somatic cell nuclear transfercells or may be present within or isolated from the retina. A retinalprogenitor cell can, in general, undergo symmetric or asymmetricdivision and thus can either differentiate into various types of retinalcells or retinal pigmented epithelial cells, or produce two furtherretinal progenitor cells. Thus, it should be understood that the cellsthat are used in the culturing step to differentiate into retinalprogenitor cells include various types of cells which were generatedduring differentiation from stem cells into retinal cells, as well asretinal progenitor cells. Retinal progenitor cells include neuralretinal progenitor cells and retinal pigmented epithelial progenitorcells and are characterized by at least one, two or three markersselected from among Rax, Pax6, Chx10, Otx2, Sox2, Lhx2, Six3, Six6, andMitf.

As used therein, the term “progenitor cell” or “precursor” refers to acell capable of asymmetric division. asymmetric division refers tosituations in which a progenitor cell or a precursor can, with a certainprobability, either produce two further progenitor or precursor cells ordifferentiate, so that although they have undergone the same rounds ofpassages, the resulting cells may have different ages and properties.

In connection with retinal development, as mentioned above, retinalprogenitor cells are able to differentiate into various types ofintraretinal cells (rod and cone photoreceptor cells, retinal ganglioncell, horizontal cells, bipolar cells, amacrine cells, Muller glialcells, etc.) and retinal pigmented epithelium, featuring positiveexpression of markers such as Crx, recoverin, rhodopsin, red/greenopsin, blue opsin, peripherin2, PDE6B, SAG, Islet1/NF200, Prox1, PKC-a,Hu C/D, GFAP, and RPE65. However, the expression level and positiveratio of these markers become weaker in retinal progenitor cells than inmature retinal cells or retinal pigmented epithelium.

As used herein, the term “neural retinal progenitor cells” is intendedto mean the retinal progenitor cells which favor neurons. That is,neural retinal progenitor cells are herein progenitor cells determinedto differentiate into intraretinal neurons (rod and cone photoreceptorcells, retinal ganglion cells, horizontal cells, bipolar cells, amacrinecells, and Muller glial cells). A neural retinal progenitor cell can, ingeneral, undergo symmetric or asymmetric division, eitherdifferentiating into various types of retinal cells or retinal pigmentedepithelial cells, or producing two further retinal progenitor cells.Thus, it should be understood that the cells in the step of culturingthat differentiate into neural retinal progenitor cells include varioustypes of cells generated during the differentiation of stem cells intoretinal cells as well as neural retinal progenitor cells. Neural retinalprogenitor cells are characterized by expressing at least one, two orthree markers selected from among Rax, Pax6, Chx10 and Crx.

In addition to expressing these markers, neural retinal progenitor cellsmay be characterized by the ability to express Crx, recoverin andrhodopsin, which are the markers of cells of the next differentiationstage, that is, photoreceptor cell precursors and photoreceptor cells.On the contrary, neural retinal progenitor cells are observed to have adecreased expression level of Otx2, Sox2, Lhx2, Six3, Six6 and Mitf,which are markers characteristic of retinal progenitor cells thatmanifest themselves in the previous differentiation stage.

As used herein, the term “retinal pigmented epithelial progenitor cell”is intended to refer to a differentiated retinal progenitor cell whichfavors retinal pigmented epithelium. Retinal pigmented epithelialprogenitor cells are characterized by expressing one or more markersselected from among Mift and Pax6.

In a preferred embodiment, differentiation from retinal progenitor cellsinto retinal cells may be achieved by (a) culturing stem cell-derivedretinal progenitor cells in a composition containing a Wnt signalingpathway activator to differentiate them into neural retinal progenitorcells; (b) culturing the neural retinal progenitor cells in acomposition containing a Wnt signaling pathway activator todifferentiate them into photoreceptor cell precursors; and (c) culturingthe photoreceptor cell precursors in a composition containing a Wntsignaling pathway activator to differentiate them into retinal cellsincluding photoreceptor cells.

As used herein, the term “photoreceptor cell precursor” means aprecursor favoring differentiation into a photoreceptor cell,characterized by one or more markers selected from among Crx (rod andcone photoreceptor cell precursors) and Nr1 (rod photoreceptor cellprecursors). In addition to expressing the markers, the photoreceptorcell precursors may also be characterized by the ability to express atleast one, two or three of recoverin, rhodopsin, peripherin2, and rom1,which are markers characteristic of differentiating photoreceptor cells.

So long as it activates Wnt signaling pathway, any Wnt signaling pathwayactivator may be used in the present invention. Examples of the Wntsignaling pathway activators useful in the present invention includeWnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b,Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16b; substancesincreasing β-catenin levels; GSK3 inhibitors such as lithium, LiCl,bivalent zinc, BIO, SB216763, SB415286, CHIR99021, QS11 hydrate, TWS119,Kenpaullone, alsterpaullone, indirubin-3′-oxime, TDZD-8 and Ro 31-8220methanesulfonate salt; Axin inhibitors, APC inhibitors, norrin andR-spondin 2, with preference for Wnt3a, Wnt1, Wnt5a, Wnt11, norrin,LiCl, BIO and SB415286.

In a preferred embodiment of the present invention, the composition forinducing the proliferation of retinal cells contains the Wnt signalingpathway activator except for LiCl, BIO and SB415286 in an amount of from0.01 to 500 ng/ml, preferably in an amount of from 0.1 to 200 ng/ml, andmore preferably in an amount of from 1 to 100 ng/ml. Among the Wntsignaling pathway activators, LiCl is used in the medium in an amount of0.1 to 50 mM, preferably in an amount of 0.5 to 10 mM, and morepreferably in an amount of 1 to 10 mM; BIO is used in an amount of 0.1to 50 μM, preferably in an amount of 0.1 to 10 μM, and more preferablyin an amount of 0.5 to 5 μM; SB415286 is used in an amount of 0.1 to 500μM, preferably in an amount of 1 to 100 μM, and more preferably in anamount of 5 to 50 μM. In a modification of the embodiment, the mediummay contain 50 ng/ml of Wnt3a or Wnt1; 50 or 100 ng/ml of Wnt5a andWnt11; 50 ng/ml of norrin; 2.5 or 5 mM of LiCl; 2 μM of BIO, or 30 μM ofSB415286. In a preferred embodiment, the composition for inducingretinal progenitor cells to differentiate into neural retinal progenitorcells in step (a) may further comprise an IGF1R activator, a BMPsignaling pathway inhibitor, and an FGF signaling pathway activator; thecomposition for inducing retinal neural progenitor cells todifferentiate into photoreceptor cell precursors in step (b) may be freeof the BMP signaling pathway inhibitor and the FGF signaling pathwayactivator and may further comprise an Shh (sonic hedgehog) signalingpathway activator; and the composition for inducing the photoreceptorcell precursors to differentiate into retinal cells includingphotoreceptor cells in step (c) may be the same as in the composition ofstep (b), with the exception that RA is further added thereto.

Any technique that is well known in the art or allows the production ofretinal progenitor cells may be employed to produce retinal progenitorcells. Preferably, the retinal progenitor cells may be obtained by: (a′)culturing stem cells in a medium containing an IGF1R activator, a Wntsignaling pathway inhibitor and a BMP signaling pathway inhibitor todifferentiate them into eye field precursors in the form of floatingaggregates; and (b′) culturing the eye field precursors in the form offloating aggregates in the same medium, but further comprising an FGFsignaling pathway activator to differentiate them into retinalprogenitor cells.

In a preferred embodiment, when cultured, the floating aggregates of eyefield precursors may be grown adhering to a plate. Any cell-adheringplate well known in the art may be employed. Preferably, it is coatedwith an extracellular matrix, such as poly-D-lysine, laminin,poly-L-lysine, matrigel, agar, polyornithine, gelatin, collagen,fibronectin or vitronectin. Most preferred is a plate coated withpoly-D-lysine/laminin. The cell population per floating aggregate whichadheres to the plate is the one that is the most highly efficient.Preferably, a floating aggregate consists of 200-400 cells.

Examples of the stem cells useful in a preferred embodiment of thepresent invention include, but are not limited to, bone marrow stemcells (BMSC), cord blood stem cells, amniotic fluid stem cells, fat stemcells, retinal stem cells (RSC), intraretinal Muller glial cells,embryonic stem cells (ESC), induced pluipotent stem cells (iPSC) andsomatic cell nuclear transfer cells (SCNTC), with the greatestpreference being for human ESC or iPSC. In an embodiment, iPSC as wellas human ESC was successfully induced to differentiate into retinalcells including photoreceptor cells by the differentiation method of thepresent invention.

As used herein, the term “eye field precursor” refers to a cellexpressing a marker (eye field transcription factors; Zuber, et al.,Development, 2003; 130: 5155-67) found in a progenitor for the eye fieldof the forebrain neural plate. The eye field precursors arecharacterized by at least one, two or three markers selected from amongSix3, Rax, Pax6, Otx2, Lhx2, and Six6.

As used herein, the term “floating aggregate” refers to a cell massfloating in a medium which is generated when a floc of stem cells iscultured for at least one day in a non-adhesive plate without feedingmouse embryonic fibroblasts and sera. Depending on the composition ofthe medium supplied, the eye field precursors may express eye fieldtranscription factors.

As used herein, the term “Wnt signaling pathway inhibitor” is intendedto refer to a factor which interrupts interaction between theextracellular Wnt protein and the membrane protein Frizzled receptor orLRP or inhibits intracellular Wnt-mediated signal transduction (Kawano &Kypta, J Cell Sci. 2003; 116: 2627-34). So long as it inhibitsWnt-mediated signal transduction, any Wnt signaling pathway inhibitormay be used in the present invention. Examples of the Wnt signalingpathway inhibitors useful in the present invention include the Dkk(Dickkopf) family (Dkk-1, Dkk-2, Dkk-3 and Dkk-4), which are Wntantagonists capable of interacting with the co-receptor LRP, Wise, thesFRP (secreted Frizzled-related protein) family, which functions as Wntantagonists binding to Wnt receptors, a Frizzled-CRD domain, WIF-1 (Wntinhibitory factor-1), IWP-2, IWP-3, IWP-4, cerberus, Wnt antibodies,dominant negative Wnt proteins, overexpression of Axin, overexpressionof GSK (glycogen synthase kinase), dominant negative TCF, dominantnegative dishevelled and casein kinase inhibitors (CKI-7, D4476 etc.),with preference for Dkk-1.

Wnt signal transduction may be inhibited by suppressing each componentinvolved in the Wnt pathway with for example RNAi, in addition to theWnt signaling pathway inhibitor.

In a preferred embodiment, IGF-1 or IGF-2 may be used as an IFG1Ractivator, with priority given to IGF-2. The medium useful for inducingthe neural retinal progenitor cells to differentiate into photoreceptorcell precursors contains IGF1R activator in an amount of from 0.01 to100 ng/ml, preferably in an amount of from 0.1 to 50 ng/ml, morepreferably in an amount of from 1 to 20 ng/ml, and most preferably in anamount of 10 ng/ml.

In a preferred embodiment, the BMP signal pathway inhibitor includenoggin, chordin, twisted gastrulation (Tsg), cerberus, coco, gremlin,PRDC, DAN, dante, follistatin, USAG-1, dorsomorphin and sclerostin, withpreference for noggin. The medium contains the BMP signaling pathwayinhibitor in an amount of from 0.01 to 100 ng/ml, preferably in anamount of from 0.1 to 50 ng/ml, more preferably in an amount of from 0.5to 20 ng/ml, and most preferably in an amount of 10 ng/ml.

As the FGF signaling pathway activator, a factor activating FGRR1c orFGFR3c, FGF1, FGF2, FGF4, FGF8, FGF9, FGF17 or FGF19 may be used, withFGF2 being preferred. The medium useful for inducing the retinalprogenitor cells to differentiate into neural retinal progenitor cellscontains the FGF signaling pathway activator in an amount of from 0.01to 100 ng/ml, preferably in an amount of from 0.1 to 50 ng/ml, morepreferably in an amount of from 1 to 20 ng/ml, and most preferably in anamount of 5 ng/ml.

In a preferred embodiment, examples of the Wnt signaling pathwayactivator useful in the present invention include Wnt1, Wnt2, Wnt2b,Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b,Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16b; substances increasingβ-catenin levels; GSK3 inhibitors such as lithium, LiCl, bivalent zinc,BIO, SB216763, SB415286, CHIR99021, QS11 hydrate, TWS119, Kenpaullone,alsterpaullone, indirubin-3′-oxime, TDZD-8 and Ro 31-8220methanesulfonate salt; Axin inhibitors, APC inhibitors, norrin andR-spondin 2, with preference for Wnt3a, Wnt1, Wnt5a, Wnt11, norrin,LiCl, BIO and SB415286. The medium useful for inducing the neuralretinal progenitor cells to differentiate into photoreceptor cellprecursors contains the Wnt signaling pathway activator except for LiCl,BIO and SB415286 in an amount of from 0.01 to 500 ng/ml, preferably inan amount of from 0.1 to 200 ng/ml, and more preferably in an amount offrom 1 to 100 ng/ml. Among the Wnt signaling pathway activators, LiCl isused in the medium in an amount of 0.1 to 50 mM, preferably in an amountof 0.5 to 10 mM, and more preferably in an amount of 1 to 10 mM; BIO isused in an amount of 0.1 to 50 μM, preferably in an amount of 0.1 to 10μM, and more preferably in an amount of 0.5 to 5 μM; SB415286 is used inan amount of 0.1 to 500 μM, preferably in an amount of 1 to 100 μM, andmore preferably in an amount of 5 to 50 μM. In a modification of theembodiment, the medium may contain 50 ng/ml of Wnt3a or Wnt1; 50 or 100ng/ml of Wnt5a and Wnt11; 50 ng/ml of norrin; 2.5 or 5 mM of LiCl; 2 μMof BIO, or 30 μM of SB415286.

In a preferred embodiment, the Shh signaling pathway activator useful inthe present invention may be Shh, an inhibitor of Ptc's interaction withSmo, an Smo receptor activator, a substance increasing Ci/Gli familylevels, an inhibitor of the intracellular degradation of Ci/Gli factors,or an Shh receptor activator (e.g. Hg—Ag, purmorphamine, etc.).Preferred is Shh or purmorphamine. In a preferred embodiment, the mediumcontains the Shh signaling pathway activator in an amount of from 0.1 to5,000 ng/ml, preferably in an amount of from 1 to 2,500 ng/ml, morepreferably in an amount of from 10 to 1,000 ng/ml, and most preferablyin an amount of 250 ng/ml. In an embodiment of the present invention,the medium contains Shh in an amount of 250 ng/ml or purmorphamine in anamount of 1 μM.

As for the period of time for culturing after differentiation starts, itis preferably given 1-30 days for step (a′), 5-15 days for step (b′),1-30 days for step (a), 1-30 days for step (b), and 1-60 days for step(c), and most preferably 4 days for step (a′), 9 days for step (b′), 5days for step (a), 3 days for step (b), and 8-15 days for step (c). In apreferred embodiment, it takes as short as approximately 29 days tocomplete the differentiation of stem cells into retinal cells, allowingthe method to be effectively applied to clinical treatment.

Differentiation into eye field precursors in step (a′) may beaccomplished by inducing and promoting the development of the forebrainduring embryogenesis with the concomitant suppression of Wnt and BMPsignaling pathways (Piccolo, et al., Nature. 1999; 397: 707-10).Therefore, the culture medium contains Dkk-1 as a Wnt signaling pathwayinhibitor, noggin as a BMP inhibitor, and IGF-1 as an IGF1R activatorfunctioning to promote the formation of the eye field in the forebrain.Kinds and medium levels of the Wnt signaling pathway inhibitor, the BMPinhibitor and IGF1R activator are as defined above.

The medium useful in step (a′) contains IGF-1 at a concentration of0.01-100 ng/ml, Dkk-1 at a concentration of 0.01-10,000 ng/ml and nogginat a concentration of 0.01-100 ng/ml, and most preferably IGF-1 at aconcentration of 5 ng/ml, Dkk-1 at a concentration of 1 ng/ml, andnoggin at a concentration of 1 ng/ml.

Any conventional medium for culturing stem cells may be used forinducing differentiation into eye field precursors in step (a′).Preferred is DMEM/F12 containing 10% knockout serum replacement, 1 mML-glutamine, 0.1 mM non-essential amino acids, 0.1 mM mercaptoethanol,and 1% B27 supplement.

Differentiation into retinal progenitor cells in step (b′) may beconducted in a medium containing an FGF signaling pathway activator,preferably FGF2, in combination with the factors of step (a′), that is,an IGF1R activator, a Wnt signaling pathway inhibitor and a BMPinhibitor.

The medium useful in step (b′) contains IGF-1 at a concentration of0.01-100 ng/ml, Dkk-1 at a concentration of 0.01-10,000 ng/ml, noggin ata concentration of 0.01-100 ng/ml and FGF2 at a concentration of0.01-100 ng/ml and most preferably IGF-1 at a concentration of 10 ng/ml,Dkk-1 at a concentration of 10 ng/ml, noggin at a concentration of 10ng/ml and FGF2 at a concentration of 5 ng/ml.

The differentiation of the retinal progenitor cells of step (b′) intoneural retinal progenitor cells in step (a), must be conducted in theabsence of Dkk-1, a Wnt signaling pathway inhibitor, for there to be ahigh level of Pax6 expressed (Pax6 is essential for the generation ofneural retinal progenitor cells), and in a medium containing Wnt3a forpromoting the Wnt pathway, noggin for converting ventral retinalpigmented epithelium into the neural retina in the development stage ofoptic vesicles and optic cups during embryogenesis, FGF2 for suppressingthe expression of genes necessary for retinal pigmented epithelium andpromoting the generation of neural retinal progenitor cells, and IGF-1responsible for the antiapoptosis of the photoreceptor cells.

The medium useful in step (a) contains IGF-1 in a concentration of0.01-100 ng/ml, noggin in a concentration of 0.01-100 ng/ml, FGF2 in aconcentration of 0.01-100 ng/ml, and Wnt3a in a concentration of0.01-500 ng/ml and most preferably IGF-1 in a concentration of 10 ng/ml,noggin in a concentration of 10 ng/ml, FGF2 at a concentration of 5ng/ml and Wnt3a at a concentration of 50 ng/ml.

Differentiation into photoreceptor cell precursors in step (b) isconducted in a medium which is free of both noggin and FGF2 thatrespectively inhibit Shh signaling pathway and Shh-induced rhodopsinexpression, and which contains IGF-1 for proliferating rod photoreceptorcell precursors, Wnt3a for promoting the Wnt pathway, and Shh.

The medium useful in step (b) contains IGF-1 in a concentration of0.01-100 ng/ml, Wnt3a at a concentration of 0.01-500 ng/ml and Shh at aconcentration of 0.1-5,000 ng/ml and most preferably IGF-1 at aconcentration of 10 ng/ml, Wnt3a at a concentration of 50 ng/ml and Shhat a concentration of 250 ng/ml.

Differentiation into photoreceptor cells in step (c) is conducted in amedium which contains RA (retinoic acid) for further promoting thedifferentiation in combination with IGF-1, Wnt3a and Shh.

The medium useful in step (c) contains IGF-1 in a concentration of0.01-100 ng/ml, Wnt3a at a concentration of 0.01-500 ng/ml, Shh at aconcentration of 0.01-5,000 ng/ml and RA at a concentration of0.5-10,000 nM and most preferably IGF-1 at a concentration of 10 ng/ml,Wnt3a at a concentration of 50 ng/ml, Shh at a concentration of 250ng/ml and RA at a concentration of 500 nM.

In steps (a) to (c), Wnt1, Wnt5a, Wnt11, norrin, LiCl, BIO or SB415286may be used instead of Wnt3a while purmorphamine may substitute for Shh.

Any conventional medium for culturing embryonic stem cells may be usedas a fundamental medium in steps (b′), (a), (b) and (c). Preferred isDMEM/F12 containing 1 mM L-glutamine, 0.1 mM non-essential amino acids,0.1 mM mercaptoethanol, 1% B27 supplement and 1% N2 supplement.

In an embodiment of the present invention, a Wnt signaling pathwayactivator was observed to play a critical role in differentiatingretinal progenitor cells into photoreceptor cells. In this regard,differentiation was conducted in the presence of various concentrationsof the Wnt signaling pathway inhibitor Dkk-1 (10 ng/ml, 100 ng/ml and 1μg/ml). In detail, hESC-derived retinal progenitor cells were induced todifferentiated into photoreceptor cells in a medium containing 50 ng/mlWnt3a, but neither Shh nor retinoic acid (group I), containing neitherWnt3a nor Dkk-1 (group II), containing 10 ng/ml Dkk-1 (group III),containing 100 ng/ml Dkk-1 (group IV) and containing 1 μg/ml Dkk-1(group V).

As a result, the photoreceptor cell markers Crx (p=0.0247), recoverin(p=0.0113), rhodopsin (p=0.0166) and peripherin2 (p=0.0166) remarkablydecreased in expression level with increasing of Dkk-1 concentration.Statistical significances were found between all groups except for groupI vs. group II and group IV vs. group V in Crx and group I vs. group IIin recoverin (significance between groups: *: p<0.0001; **: p=0.0381)(Table 10 and FIG. 24). These data showed that the differentiation intophotoreceptor cells is induced by a Wnt signaling pathway activator andthat the inhibition of the Wnt signaling pathway leads to almost nogeneration of photoreceptor cells, giving evidence of the Wnt signalingpathway inhibitor playing a critical role in differentiation intophotoreceptor cells.

On the other hand, respective substitutes may be used instead of Wnt3aand Shh. In this context, the culturing processes are carried out forthe same time period under the same conditions with the exception that50 ng/ml of Wnt1; 50 and 100 ng/ml of Wnt5a and Wnt11; 50 ng/ml ofnorrin; 2.5 or 5 mM of LiCl; 2 μM of BIO; or 30 μM of SB415286 is usedas a Wnt signaling pathway activator in substitution for Wnt3a andpurmorphamine is used as a substitute for Shh. These substitutes, as aresult, give rise to the differentiation and maturation of photoreceptorcells in a similar pattern to that obtained with Wnt3a and Shh (Example12).

MODE FOR INVENTION

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as limiting the present invention.

Example 1 Culture of Stem Cells

<1-1> Culture of Human Embryonic Stem Cells

The human embryonic stem cell (hESC) lines H9 (WA09, normal karyotypeXX) and H7 (WA07, normal karyotype, XX) were purchased from the WiCellResearch Institute (Madison, Wis., USA).

The hESCs were allowed to proliferate undifferentiated (H9 cells:passages 25^(˜)33; H7 cells: passages 23^(˜)28) by culturing over feedercells, such as radiated mouse embryonic fibroblasts (MEF, ATCC,Manassas, Va., USA) or mitomycin-treated mouse embryonic fibroblasts(EmbryoMax Primary Mouse Embryo Fibroblasts, Millipore, Billerica,Mass., USA) in the following medium: DMEM/F12 (Invitrogen, Grand Island,N.Y., USA), 20% (v/v) KnockOut serum replacement, Invitrogen, Carlsbad,Calif., USA), 1 mM L-glutamine (Invitrogen), 0.1 mM nonessential aminoacids (Invitrogen), 0.1 mM mercaptoethanol (Sigma-Aldrich, St. Louis,Mo., USA), and 4 ng/ml human recombinant basic fibroblast growth factor(FGF2, Invitrogen).

While the medium was replaced with a fresh one every day, theundifferentiated stem cells were passaged at a ratio of 1:9-1:15 everysix or seven days manually or with collagenase IV (Invitrogen), and thentransferred onto fresh MEF feeder cells. During the passage of thehESCs, immunochemical staining with OCT-4 and SSEA-4 (Chemicon,Temecula, Calif., USA), antigens characteristic of undifferentiatedhESCs, was conducted at regular intervals of time to monitor the degreeof differentiation. Cells that were found to have undergodifferentiation were removed.

The presence of mycoplasma contamination in the hESC culture, whichcould have an undesirable effect on the differentiation of hESCs, wasregularly monitored with a kit (MycoAlert mycoplasma detection kit,Lonza, Rockland, Me., USA).

<1-2> Culture of Induced Pluipotent Stem Cells (iPSCs)

The human iPSC line iPS (Foreskin)-1 (Clone 1) (normal karyotype XY) waspurchased from the WiCell Research Institute (Madison, Wis., USA).

The human iPSCs were allowed to proliferate undifferentiated (passages37-47) by culturing them over feeder cells, such as radiated mouseembryonic fibroblasts (MEF, ATCC, Manassas, Va., USA) ormitomycin-treated mouse embryonic fibroblasts (EmbryoMax Primary MouseEmbryo Fibroblasts, Millipore, Billerica, Mass., USA) in the followingmedium: DMEM/F12 (Invitrogen, Grand Island, N.Y., USA), 20% (v/v)KnockOut serum replacement (Invitrogen, Carlsbad, Calif., USA), 1 mML-glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen),0.1 mM mercaptoethanol (Sigma-Aldrich, St. Louis, Mo., USA), and 10ng/ml human recombinant FGF2 (Invitrogen).

While the medium was replaced with a fresh one every day, theundifferentiated stem cells were passaged at a ratio of 1:4-1:6 everysix or seven days manually or with collagenase IV (Invitrogen), and thentransferred onto fresh MEF feeder cells. During the passage of thehESCs, immunochemical staining with SSEA-4 (Chemicon, Temecula, Calif.,USA) and Nanog (abcam, Cambridge, Mass., USA), which are antigenscharacteristic of undifferentiated human iPSCs, was conducted at regularintervals of time to monitor the degree of differentiation. Cells thatwere identified to have undergone differentiation were removed.

The presence of mycoplasma contamination in the hESC culture, whichcould have an undesirable effect on the differentiation of hESCs, wasregularly monitored with a kit (MycoAlert mycoplasma detection kit,Lonza, Rockland, Me., USA).

Example 2 Differentiation from hESCs or Human iPSCs into Eye FieldPrecursors

The hESCs or human iPSCs cultured in Example 1 were separated from theMEF cells (FIGS. 1 and 14) and seeded into 6-well ultra-low attachmentplates (Corning Incorporated, Corning, N.Y., USA).

To the hESCs or human iPSCs in the 6-well ultra-low attachment plateswas added a medium for inducing differentiation into eye fieldprecursors [DMEM/F12, 10% KnockOut serum replacement, 1 mM L-glutamine,0.1 mM nonessential amino acids, 0.1 mM mercaptoethanol, 1% B27supplement (Invitrogen), 1 ng/ml recombinant noggin (R&D Systems), 1ng/ml recombinant Dkk-1 (Dickkopf-1, R&D Systems), and 5 ng/mlrecombinant IGF-1 (insulin-like growth factor-1, R&D Systems)]. Thecells were cultured for 4-5 days to generate eye field precursors in theform of floating aggregates with the replacement of the medium with afresh one every third day (FIG. 1).

Example 3 Differentiation from Eye Field Precursors into RetinalProgenitor Cells

The eye field precursors (floating aggregates) generated in Example 2were seeded at a density of 53±8 cells per well (292±53 cells/floatingaggregate) into 6-well poly-D-lysine/laminin-coated plates (BDBiosciences) and at a density of 12±4 cells per well on 8-wellpoly-D-lysine/laminin-coated plates and then cultured for 9 days togenerate retinal progenitor cells, with the supply of a medium forinducing differentiation into retinal progenitor cells [DMEM/F12(Invitrogen), 1 mM L-glutamine (Invitrogen), 0.1 mM nonessential aminoacids (Invitrogen), 0.1 mM mercaptoethanol (Sigma-Aldrich), 1% B27supplement, 1% N2 supplement (Invitrogen), 10 ng/ml Dkk-1, 10 ng/mlnoggin, 10 ng/ml IGF-1, and 5 ng/ml FGF2].

Example 4 Differentiation from Retinal Progenitor Cells into NeuralRetinal Progenitor Cells

To differentiate the retinal progenitor cells generated in Example 3into neural retinal progenitor cells, Dkk-1 supply was quit on day 3after induction and a culture medium containing a combination of noggin,IGF-1, FGF2 and Wnt3a was supplied for 5 days (FIG. 1). The culturemedium was composed as follows: [DMEM/F12 (Invitrogen), 1 mM L-glutamine(Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mMmercaptoethanol (Sigma-Aldrich), 1% B27 supplement (Invitrogen), 1% N2supplement (Invitrogen), 10 ng/ml noggin, 10 ng/ml IGF-1, 5 ng/ml FGF2,and 50 ng/ml recombinant Wnt3a (R&D Systems)].

Example 5 Differentiation from Neural Retinal Progenitor Cells intoPhotoreceptor Cell Precursors

The neural retinal progenitor cells generated in Example 4 were inducedto differentiate into photoreceptor cell precursors by supplying amedium [DMEM/F12 (Invitrogen), 1 mM L-glutamine (Invitrogen), 0.1 mMnonessential amino acids (Invitrogen), 0.1 mM mercaptoethanol(Sigma-Aldrich), 1% B27 supplement (Invitrogen), 1% N2 supplement(Invitrogen), 10 ng/ml IGF-1, 50 ng/ml Wnt3a, and 250 ng/ml recombinantShh (Sonic Hedgehog amino terminal peptide, Shh, R&D Systems)] for 3days. Noggin and FGF2, both used in the previous step, were excludedfrom the medium (FIG. 1).

Example 6 Differentiation from Photo Receptor Cell Precursors into PhotoReceptor Cells

A specialized medium [DMEM/F12 (Invitrogen), 1 mM L-glutamine(Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mMmercaptoethanol (Sigma-Aldrich), 1% B27 supplement (Invitrogen), 1% N2supplement (Invitrogen), 10 ng/ml IGF-1, 50 ng/ml Wnt3a, 250 ng/ml Shh,500 nM (all-trans retinoic acid (RA, Sigma-Aldrich)] were supplied for 8days or longer to induce the photoreceptor cell precursors todifferentiate into photoreceptor cells.

All the media were replaced with fresh ones every two or three days inExamples 2 to 6, and the cells were cultured at 37° C. in a 5% CO₂atmosphere. All induction and differentiation experiments were repeatedat least three times and the same results were obtained therefrom.

Example 7 Assay for Cellular Differentiation-Related Markers

<7-1> Immuno Chemical Staining and Identification of CellularDifferentiation-Related Marker Expression

The differentiation of the cells obtained in Examples 3 to 6 wasexamined using an immunochemical staining method as follows.

The eye field precursors (floating aggregates) were cultured in 8-wellpoly-D-lysine/laminin-coated slides (BD Biosciences, Bedford, Mass.)under the same conditions that were used for differentiation into theretinal progenitor cells, the neural retinal progenitor cells, thephotoreceptor cell precursors and the photoreceptor cells. The cellscompletely cultured in each step were fixed with 4% paraformaldehyde(Sigma-Aldrich), after which non-specific reactions were blocked withPBS containing 3% BSA (Jackson Immunoresearch Laboratory, Bar Harbor,Me., USA) and 0.25% Triton X-100 (Sigma-Aldrich).

After being blocked for 90 min, the slides in each differentiation stepwere incubated overnight at 4° C. with the following antibodies specificfor cells of each differentiation step: rabbit-Blue-opsin (1:500,Chemicon), sheep-Chx10 (1:100, Chemicon), rabbit-Crx (1:200, Santa CruzBiotechnology, Santa Cruz, Calif., USA), rabbit-GFAP (1:200,Invitrogen), mouse-human specific mitochondria (1:50, Chemicon),rabbit-human specific mitochondria (1:200, Chemicon), mouse-Islet1(1:10, Developmental Studies Hydroma Bank, DSHB; Iowa City, Iowa, USA),mouse-Ki67 (1:100, Vector Laboratories, Peterborough, England),mouse-Mitf (1:1,000, abcam, Cambridge, Mass., USA), mouse-nestin (1:250,BD Sciences), rabbit-neurofilament-200 (1:1,000, Sigma-Aldrich),rabbit-Otx2 (1:100, abcam), rabbit-Pax2 (1:250, abcam), mouse-Pax6 (1:2,DSHB), mouse-peripherin2 (1:500, GenScript, Piscataway, N.J., USA),rabbit-Rax (1:250, abcam), rabbit-recoverin (1:1,000, Chemicon), retinalpigment epithelium 65 (RPE65, 1:100, Chemicon), rabbit-rhodopsin (1:500,Sigma-Aldrich), mouse-rhodopsin (1:500, Ret-P1, Lab Vision, Fremont,Calif., USA), mouse-rhodopsin (1:2,000, Ret-P1, Sigma-Aldrich),mouse-rom1 (1:50, ABR-Affinity Bioreagents, Golden, Colo., USA),rabbit-PDE6 beta (1:100, abcam), rabbit-phosducin (1:500, Santa CruzBiotechnology), mouse-PKC-alpha (1:500, Sigma-Aldrich), mouse-Prox1(1:2,000, Chemicon), mouse-Sox2 (1:250, R&D Systems),rabbit-synaptophysin (1:2,000, Santa Cruz Biotechnology), andrabbit-ZO-1 (1:100, Zymed-Invitrogen).

Before use, these antibodies were diluted in a PBS solution containing1% BSA and 0.25% Triton X-100. The cells cultured on the slides in eachstep were washed three times for 5 min with PBS and incubated at roomtemperature for 2 hrs with species-specific secondary antibodyconjugated with Cy3 (1:800, Jackson Immunoresearch Laboratory) orAlexa488 (1:500, Invitrogen). A standard material suitable for theprimary and the secondary antibody was used to examine non-specificstaining or interaction between the antibodies. Afterwards, the cellswere washed three times for 5 min with PBS, counterstained with DAPI(4,6-diamidino-2 phenylindole) and mounted in Vectashield (VectorLaboratories), followed by visualization under an epifluorescencemicroscope (Nikon Eclipse, E800, Tokyo, Japan) and a confocal microscope(Leica, Leica Microsystems Inc, Bannockburn, Ill., USA or Zeiss LSM510,Carl Zeiss, Inc, Thornwood, N.Y., USA).

500 cells were counted from 20 microscopic fields randomly selected at200 magnification and evaluated for positive responses to each antibody.Positive responses to antibodies were determined after at least threeevaluations. Statistical analysis of the data was done using theKruskal-Wallis test and the Bland-Altman plot (Bland and Altman, 1986)of Med Calc version 8.1.1.0 as well as the GEE (Generalized EstimatingEquations) model of SAS version 9.1. All data were represented asmean±standard error of the mean (S.E.M) with a statistical significanceof p<0.05.

With regard to the markers characteristic of both forebrain eye fieldprecursors and retinal progenitor cells, Rax was found to be expressedon the retinal progenitor cells generated in Example 3 at a positiverate of 86.6±3.0%, Pax6 at a positive rate of 63.9±0.9%, Otx2 at apositive rate of 76.4±2.0%, Sox2 at a positive rate of 83.0±1.9%, andChx10 at a positive rate of 46.3±1.0%. The cells were also found toexpress the marker Mitf characteristic of retinal pigmented epithelialprogenitor cells at a positive rate of 17.2±0.4%, and the marker nestincharacteristic of neural progenitor cells at a positive rate of65.7±2.7% (Tables 1 and 2).

TABLE 1 Change in Marker Level with Culture Time Period Mean ± S.E.M.(%, Sample size = 3) Marker 13 Days 18 Days 21 Days 30 Days Rax86.6(±3.0) 98.2(±0.9) 97.6(±0.6) 97.8(±0.7) Pax6 63.9(±0.9) 89.1(±2.5)61.6(±0.5) 89.7(±1.9) Otx2 76.4(±2.0) 61.5(±0.6) 63.3(±2.9) 57.2(±2.4)Sox2 83.0(±1.9) 68.1(±1.1) 49.1(±1.6) 69.1(±4.0) Chx10 46.3(±1.0)64.5(±1.6) 48.5(±3.9) 41.6(±2.0) Nestin 65.7(±2.7) 18.0(±1.4) 14.3(±2.3) 8.5(±0.9) Mitf 17.2(±0.4)  2.7(±1.0) 18.6(±1.3) 24.7(±0.3)

On the neural retinal progenitor cells generated in Example 4, thepositive rates were increased simultaneously from 63.9% to 89.1% for Pax6, from 86.6% to 98.2% for Rax, and from 46.3% to 64.5% for Chx10(p<0.0001), indicating that the increase in the positive rate of Pax6resulted from the proliferation of neural retinal progenitor cells (bothRax+ and Pax6+ positive) (Tables 1 and 2).

TABLE 2 Statistical Significance of Retinal Cell Markers between CulturePeriods of Time Statistical Significance (p values) 13 Days vs. 18 Daysvs. 21 Days vs. Marker 18 Days 21 Days 30 Days Rax <0.0001 0.5308 0.7897Pax6 <0.0001 <0.0001 <0.0001 Otx2 <0.0001 0.4596 0.0505 Sox2 <0.0001<0.0001 <0.0001 Chx10 <0.0001 <0.0001 0.0531 Nestin <0.0001 0.10770.0010 Mitf <0.0001 <0.0001 0.0134 *Statistical significance: p < 0.05

As shown in Tables 1 and 2, the positive rate of the neural progenitorcell marker nestin greatly decreased from 65.7% to 18.0% (p<0.0001),from which it is understood that on differentiation day 18, theproliferation of neural progenitor cells was restrained while theproliferation and differentiation of neural retinal cells were promoted.

On the other hand, the proliferative cell marker Ki67 rapidly decreasedfrom 87.5% to 31.5% (p<0.0001), indicating that differentiation was moreactive than proliferation (FIG. 2 and Tables 3 and 4).

TABLE 3 Change in Expression Level of Retinal Cell Markers with CulturePeriod of Time Mean ± S.E.M. (%, sample size = 3) Marker 13 Days 18 Days21 Days 30 Days Crx 12.8(±1.6) 80.1(±0.2) 54.8(±4.2) 39.5(±7.4)Recoverin 35.8(±0.4) 68.5(±2.6) 61.3(±1.8) 82.4(±4.6) Rhodopsin35.3(±1.7) 52.9(±3.4) 60.4(±3.2) 81.2(±2.5) Peripherin2  5.6(±0.3)13.9(±2.4) 26.0(±0.4) 41.2(±2.0) Ki67 87.5(±1.3) 31.5(±0.5) 58.4(±4.1)30.2(±6.1)

TABLE 4 Statistical Significance of Retinal Cell Markers between CulturePeriods of Time Statistical Significance (p values) 13 Days vs. 18 Daysvs. 21 Days vs. Marker 18 Days 21 Days 30 Days Crx <0.0001 <0.00010.0326 Recoverin <0.0001 0.0079 <0.0001 Rhodopsin <0.0001 0.0489 <0.0001Peripherin2 <0.0001 <0.0001 <0.0001 Ki67 <0.0001 <0.0001 <0.0001*Statistical significance: p < 0.05

The positive rate of the photoreceptor cell precursor marker Crx showeda drastic increase from 12.8% before Wnt3a addition to 80.1% after Wnt3aaddition (p<0.0001) (FIG. 2 and Tables 3 and 4). An increase in theexpression level from 35.8% to 68.5% (p<0.0001) was also found in theuniversal photoreceptor cell marker recoverin, in the rod photoreceptorcell marker rhodopsin from 35.3% to 52.9% (p<0.0001), and in thephotoreceptor cell's outer segment marker peripherin2 from 5.6% to 13.9%(p<0.0001) (FIG. 2, Tables 3 and 4). Mitf, an antigen marker of earlypigmented epithelial progenitor cells (Baumer, et al., Development.2003; 130: 2903-15), showed a decrease in positive rate from 17.2% to2.7% (p<0.0001) (Tables 1 and 2). Also, a decrease in positive rate wasfound in the retinal progenitor cell markers Otx2 (p<0.0001) and Sox2(p<0.0001), implying differentiation into neural retinal progenitorcells.

In the photoreceptor cell precursors generated in Example 5, decreasedexpression levels were detected for the markers of both retinalprogenitor cells and neural retinal progenitor cells, including Pax6(from 89.1% to 61.6%, p<0.0001), Chx10 (from 64.5% to 48.5%, p<0.0001)and Sox2 (from 68.1% to 49.1%, p<0.0001) (Tables 1 and 2). Thephotoreceptor cell precursor marker Crx also decreased from 80.1% to54.8% (p<0.0001) (FIG. 2, Tables 3 and 4). On the other hand, anincrease in expression level was detected in the photoreceptor cellmarker rhodopsin (from 52.9% to 60.5%, p=0.0489) and in thephotoreceptor cell's outer segment marker peripherin2 (from 13.9% to26.0%, p<0.0001), indicating the differentiation and maturation ofphotoreceptor cells (FIG. 2, Tables 3 and 4). Further, the decreasingpositive rate of Ki67 was reversed into increasing from 31.5% to 58.4%(p<0.0001) (FIG. 2, Tables 3 and 4), from which it is understood thatShh started to promote cell proliferation.

As high as 82.4±4.6% of the population of cells generated by the methodof Example 6 for differentiation into photoreceptor cells were found toreact positive to recoverin, a universal marker of photoreceptor cells(rod photoreceptor cells and cone photoreceptor cells), as measured by aquantitative antigen assay (FIG. 3). Also, the quantitative antigenassay showed that 81.2±2.5% of the cell population was positive torhodopsin, an antigen characteristic of rod photoreceptor cells (FIG.3). The fact that almost all of the rhodopsin-positive cells show apositive reaction to recoverin ensures the reliability of the positiveresponses.

More precise determination of the existence and integrity of therhodopsin molecules formed by the method of the present invention wascarried out using human-specific recombinant rhodopsin (consisting ofthe amino acids of the second extracellular loop in human rhodopsin) andRet-P1 (consisting of N-terminal amino acids at positions 4 to 10 inrhodopsin), both of which are rhodopsin antibodies which recognizedifferent epitopes.

The human-specific recombinant rhodopsin and the Ret-P1 were found tohave identical positive rates, with a statistically significantdifference therebetween, as measured by the Bland-Altman plot (averageof difference in positive rate between the two antibodies: −1.00, 95%confidence interval: −13.6−11.6). The cells reacted with almost nodifference in the positive rate with both human-specific recombinantrhodopsin and Ret-P1 antibodies. This implies that the rhodopsinmolecule formed according to the method of the present invention retainstwo different epitopes therein.

The rod photoreceptor cell's outer segment markers peripherin2 (Prph2)and rom1 (retinal outer segment membrane protein 1) were positivelydetected in 41.2±2.0% and 76.0±4.6% of the population of the cellscultured, respectively (FIG. 4). Both of these two markers were observedonly in rhodopsin-positive cells (FIG. 4). Positive rates in thephotoreceptor cells were measured to be 49.8±2.2% for phosducin, whichresponds to light, that is, participates in the regulation of visualphototransduction (FIG. 5) and 43.0±2.0% for synaptophysin which isresponsible for synaptic interactions with other intraretinal neurons(FIG. 6). These facts prove that these cells participate in the neuralcircuits of the retina and perform the function of transducing lightstimuli into neural electric stimuli. Taken together, the data obtainedabove demonstrate that the method of the present invention candifferentiate with high efficiency human embryonic stem cells intophotoreceptor cells.

Blue opsin-cone photoreceptor cells were observed in 80.2±0.6% of allthe cells (FIG. 7). These cells were found from rhodopsin-positive cellflocs or in the vicinity thereof, some of which were positive to bothrhodopsin and blue opsin. Accordingly, the method of the presentinvention was proven capable of inducing human embryonic stem cells todifferentiate into both rod and cone photoreceptor cells.

Of the total population of the cells, 39.5±7.4% were positive to Crx, amarker characteristic of photoreceptor cell precursors, and 30.2±6.1%were positive to Ki67, a nuclear marker of proliferating cells (late G1phase to M phase in the cell cycle). Photoreceptor cell precursors arereported to express Crx immediately after leaving the cell proliferationcycle. Also in the present invention, most of the human ESC-derived,Crx-positive cells were observed to be negative to K167, a markerassociated with cell proliferation. However, the Crx-positive cells werefound to express Ki67 at a positive rate of 5.5±1.7% (FIG. 9).

From the data obtained above, it is apparent that the method of thepresent invention can effectively differentiate photoreceptor cells fromhuman ESC-derived retinal progenitor cells in the same multi-stepinduction pattern as they do from embryonic retinal progenitor cells,where differentiation progresses in steps along the early and lateembryonic stage. The retinal progenitor cell marker Pax6 had a positiverate of 89.7±1.9% and was detected in amacrine cells, which are a typeof differentiated retinal neurons. Meanwhile, the retinal progenitorcells expressed Mitf (a marker of retinal pigmented epithelium (RPE)progenitor cells) at a positive rate of 24.7±0.3%, and ZO-1 (a marker ofdifferentiated RPE) at a positive rate of 12.5±1.4%, and were alsopositive to RPE65, a marker of differentiated RPE (FIG. 10).

<7-2> RT-PCR and RNA mRNA Expression of CellularDifferentiation-Associated Markers

To examine the differentiation of the photoreceptor cells generated inExample 6, a reverse transcriptase-polymerase chain reaction (RT-PCR)was performed on Day 29 of differentiation induction.

Total RNA was isolated with an RNeasy minikit (Qiagen, Valencia, Calif.,USA) and DNA was removed from the RNA isolate using DNaseI (AppliedBiosystems/Ambion, Austin, Tex., USA). 500 g of the RNA was reversetranscribed into cDNA in the presence of reverse transcriptase(Omniscript reverse transcriptase, Qiagen), with random hexamers(Invitrogen) serving as primers. For the PCR, 1×PCR buffer, 1.5 mMMgCl₂, 0.2 mM dNTPs, 50 ng DNA, 0.5 U AmpliTaq Gold (Applied Biosystems,Foster City, Calif., USA) and 10 pmoles of each of the primers given inTable 5, below, were used.

TABLE 5 Forward and Reverse Primer Sequences for RT-PCR SEQ PCR IDPrimer Sequence Product Gene NO. (5′ to 3′) Size (bp) PAX6 1 Foward: 275aacagacacagccctcacaaaca PAX6 2 Reverse: cgggaacttgaactggaactgac SIX3 3Foward: 307 cgggagtggtacctacagga SIX3 4 Reverse: ttaccgagaggatggaggtgSIX6 5 Foward: 272 cctgcaggatccatacccta SIX6 6 Reverse:tgatggagatggctgaagtg LHX2 7 Foward: 285 ccaaggacttgaagcagctc LHX2 8Reverse: tgccaggcacagaagttaag RAX 9 Foward: 495 ggcaaggtcaacctaccaga RAX10 Reverse: gtgctccttggctttcagac CRX 11 Foward: 353 gtgtggatctgatgcaccagCRX 12 Reverse: tgagatgcccagagggtct Chx10 13 Foward: 281ggcgacacaggacaatcttt Chx10 14 Reverse: atccttggctgacttgagga NRL 15Foward: 206 ggcactgaccacatcctctc NRL 16 Reverse: ggaggcactgagctgtaaggRCVRN 17 Foward: 150 agctccttccagacgatgaa RCVRN 18 Reverse:caaactggatcagtcgcaga RHO 19 Foward: 258 taagcccatgagcaacttcc RHO 20Reverse: agctgcccatagcagaaaaa RBP3 21 Foward: 291 cagcccatatccctgagaatRBP3 22 Reverse: agcacaagatgggaatggag PDE6B 23 Foward: 409aggagaccctgaacatctacc PDE6B 24 Reverse: atgaagcccacttgcagc OPN1SW 25Foward: 206 ctgggcactgtagcaggtct OPN1SW 26 Reverse: tgcaggccctcagggatgASCL1 27 Foward: 467 catctcccccaactactcca ASCL1 28 Reverse:cttttgcacacaagctgcat NEUROD1 29 Foward: 523 gccccagggttatgagactatcact NEUROD1 30 Reverse: ccgacagagcccagatgtagttctt ATHO7 31 Foward: 246tcgcatcatcagacctatgg ATHO7 32 Reverse: ccgaacaggacaaactcaca POU4F2 33Foward: 175 caaccccaccgagcaata POU4F2 34 Reverse: gtgcacgggatggtattcatARX 35 Foward: 462 tgaaacgcaaacagaggcgcta ARX 36 Reverse:tgatgaaagctgggtgtcggaaca AFP 37 Foward: 318 tttagctgacctggctaccat AFP 38Reverse: cagcttgtgacaggttctgg T 39 Foward: 541 ccgtctccttcagcaaagtc T 40Reverse: caattgtcatgggattgcag GAPDH 41 Foward: 302 agccacatcgctcagacaccGAPDH 42 Reverse: gtactcagcgccagcatcg SAG 43 Foward: 400aaaaagtgccaccaaacagc SAG 44 Reverse: acgtcattcttgtctctcttcc 44 Reverse:acgtcattcttgtctctcttcc

After initial DNA denaturation at 94° C. for 10 min, all PCR wasperformed over 35 cycles starting with initial DNA denaturation (94° C.for 30 sec, 60° C. for 30 sec, 72° C. for 1 min) and terminating withextension at 72° C. for 10 min. The PCR products thus obtained wereisolated by electrophoresis on 2% agarose gel and analyzed.

All experiments were conducted in triplicate and human GAPDH was used asa reference molecule for standard mRNA calculation.

The retinal progenitor cell-related genes RAX, PAX6, SIX3, SIX6, LHX2and Chx10 were detected in the RT-PCR products. Inter alia, RAX and PAX6were expressed to as the same high extent as was the quantitativecontrol gene GAPDH. Genes relevant to photoreceptor cells and otherretinal cells were also examined for mRNA expression. The PCR productswere observed to include the photoreceptor cell-associated genes CRX,NRL, RCVRN, RHO, PDE6B, SAG and OPN1SW, the retinal ganglioncell-related genes ATHO7 and POU4F2, and the amacrine cell-related geneNEUROD1, and the bipolar cell-related gene ASCL1 (FIG. 16).

<7-3> Base Sequencing for Identifying mRNA Expression of Recoverin andRhodopsin, Both Specific for Differentiated Photoreceptor Cells

In order to investigate the differentiation of the photoreceptor cellsgenerated in Example 6, base sequencing was conducted on thephotoreceptor cell-specific genes recoverin (RCVRN: NM_(—)002903.2) andrhodopsin (RHO: NM_(—)000539.3) on Day 29 of differentiation.

The PCR products were purified with a QIAquick 96-well PCR purificationkit (Qiagen, Valencia, Calif.) and sequenced using the forward andreverse primers listed in Table 5, with the aid of a base sequencing kit(BigDye terminator cycle sequencing ready reaction kit, AppliedBiosystems, Foster City, Calif., USA). The fluorescent labeled fragmentsthus produced were purified by ethanol precipitation, resuspended indistilled water, and electrophoresed in an ABI PRISM 3700 DNA analyzer(Applied Biosystems, Foster City, Calif., USA), followed by analysis ofthe base sequences with a sequencer (Gene Codes Corporation, Ann Arbor,Mich., USA).

The RCVRN and RHO genes expressed in the photoreceptor cellsdifferentiated according to the present invention were found toperfectly coincide with human standard sequences(http://www.ncbi.nlm.nih.gov), indicating that the photoreceptor cellsexpress human RCVRN and RHO genes (FIG. 17).

Example 8 Transplantation of Photoreceptor Cells Differentiated fromhESCs and Evaluation of Transplanted Cells

<8-1> Transplantation of Differentiated Photoreceptor Cells

The transplantability and clinical applicability of the photoreceptorcells generated in Example 6 to blind persons were examined on theimmunosuppressant retinal-degeneration mouse model rd/SCID.

After getting permission from the institutional Review Board of theSeoul National University College of Medicine/the Seoul NationalUniversity Hospital and the Institutional Animal Care and Use Committee(IACUC) of Seoul National University/the Seoul National UniversityHospital, all animal experiments were conducted according to the ARVOStatement for the Use of Animals in Ophthalmic and Vision Research. Forthe transplantation, four-week-old C3H/Prkdc mice (rd/SCID, JacksonLaboratory, BarHarbor, Me., USA) were employed. C3H/Prkdc mice were alsoused as a negative control.

The retinal cells comprising photoreceptor cells differentiated fromhESCs according to the present invention were detached with accutase andsuspended in Dulbecco's PBS (D-PBS, Invitrogen) to form a single-cellsuspension with a density of 6-10×10⁴ cells/μL. A surgical procedure wascarried out under a dissecting microscope (SZ51, Olympus, Tokyo, Japan).After the mice were anesthetized by intrapeneal injection of a mixtureof zoletil (7.5 mg/kg, Virbac Laboratoires, Carros, France) and xylazine(10 mg/kg, BayerKorea, Korea), drops of a cycloplegic-mydriatic agent(Mydrin-P, Santen Pharmaceutical Co., Osaka, Japan) and 0.5%proparacaine (Alcaine 0.5%, Alcon, Inc., Puurs, Belgium) were put intothe eyes. 1 μL of the cell suspension was transplanted into each mouseby subretinal injection using a 10 μL injector (NanoFil microsyringe,World Precision Instruments, WPI, Sarasota, Fla., USA) equipped with a35-gauge needle (WPI).

<8-2> Electroretinography Retinal Degeneration after Transplantationinto Mice with Retinal Degeneration

The retinal function of the mice transplanted with the photoreceptorcells differentiated from hESCs according to the present invention wasevaluated by electroretinography (ERG, Roland Consult, Wiesbaden,Germany) 3-5 weeks after transplantation. For this, the mice wereacclimated to darkness for one day before the test. Prior to theelectroretinography, the mice were anesthetized by intraperitonealinjection of a mixture of zoletil (7.5 mg/kg, Virbac Laboratoires,Carros, France) and xylazine (10 mg/kg, BayerKorea, Korea) and drops ofMydrin-P (Santen Pharmaceutical Co., Osaka, Japan) and 0.5% proparacaine(Alcaine 0.5%, Alcon, Inc., Puurs, Belgium) were applied to the eyes.Simultaneously, vidisic Gel (Dr. Mann Parma, Berlin, Germany) was alsoput into the eyes so as to prevent eye dryness.

While the body temperature of the mice was maintained at 37° C. on amouse table (Roland Consult, Wiesbaden, Germany), the responses ofretinal nerves to light at intensities of −25, −10, and 0 dB wererecoded with the RETIport System (Roland Consult, Wiesbaden, Germany),and the records were analyzed with the RETI-scan System (Roland Consult,Wiesbaden, Germany). The same electroretinography was performed onnon-transplanted C57BL6 mice for positive control and onnon-transplanted C3H/Prkdc mice (rd/SCID) for negative control.

After the retinoelectrography, the mice were subjected to euthanasia byCO₂ injection and cervical dislocation. The eyeballs were excised,followed by immunofluorescent staining. In detail, the eyeballs excisedimmediately after euthanasia were fixed overnight at 4° C. with 4%paraformaldehyde, cryoprotected in 10% and 30% sucrose (Sigma-Aldrich),embedded in OCT (Tissue-Tek, Sakura, Tokyo, Japan), and immediatelystored at −80° C. Four weeks after the transplantation, some of the micewere subjected to euthanasia by CO₂ injection and cervical dislocationwithout electroretinography before the excision of the eyeballs. Then,immunofluorescent staining was performed on the eyeballs as describedabove.

Recovered signals in the electroretinograms reflect the effects andefficacies of the cells transplanted into injured retinas. Particularly,the existence of rhodopsin within the retina of the photoreceptorcell-transplanted mice is closely associated with the amplitude ofb-waves. In the electroretinography, non-transplanted rd/SCID mice ofthe same age (7-9 weeks old) did not respond to light stimuli (FIG.18A). In contrast, photoreceptor cell-transplanted rd/SCID mice gavedefinite responses to light stimuli (FIG. 18B). The ERG b-wave from theeyes of the photoreceptor cell-transplanted rd/SCID mice showedcharacteristic wave forms (FIG. 18B) with an amplitude of 48.4(±3.4) μV(sample size=13) (FIG. 19). Nowhere were characteristic wave forms foundin the ERG of the non-transplanted group, which showed a b-waveamplitude of 10.3 (±2.5) μV (sample size=17) different from that of thetransplanted group with statistical significance (p<0.0001) (Table 6,FIG. 19).

TABLE 6 Electroretinography of Mice with Retinal Degeneration AfterTransplantation with hESC-Derived Photoreceptor Cells Non-TransplantedGroup Transplanted Group (Mean ± S.E.M.) (Mean ± S.E.M.) (Sample Size =17) (Sample Size = 13) b-Wave 10.3(±2.5) 48.4(±13.4) Amplitude (μV)<8-3> Immunochemistry and Evaluation of Transplanted Cells

The cryoprotected segments were sectioned at a thickness of 16 μm andfixed onto glass slides (Muto Pure Chemicals Co., Tokyo, Japan),followed by immunochemical staining with antibodies to humanmitochondria and photoreceptor cells (no. of mouse models=13).

When the photoreceptor cells were transplanted into the retina of themice suffering from retinal degeneration, a new 4- or 5-foldphotoreceptor cell layer was formed, comprising the outer nuclear layerconsisting of cells characterized by rhodopsin and recoverin (FIG. 20).There was a remarkable difference in the positive rate of rhodopsin andrecoverin between photoreceptor cell-transplanted group andnon-transplanted cells. In the non-transplanted group, rhodopsin wasdetected in only two of a total of 199 cells per observation microscopicfield (positive rate: 1.0%). In contrast, 88 of a total of 215 cells permicroscopic field were rhodopsin positive in the transplanted group,with an outer nuclear layer consisting of a 4- or 5-foldrhodopsin-positive cell layer formed therein (positive rate: 40.8%)(p<0.0001) (Table 7 and FIG. 21).

As a result, the transplanted rod photoreceptor cells were found tooccupy approximately 40% of the total area of the retinal sections.Rhodopsin discs, which are embedded in the membrane of the outer segmentof photoreceptor cells in the normal retina, were also found in thephotoreceptor cells of the transplanted mice (FIG. 20). Primarilystructured to convert light energy into electric energy, rhodopsin discsare responsible for the first events in the perception of light. Theengrafted/differentiated photoreceptors of the transplanted mice,particularly, the rhodopsin discs of the outer segment were thought toinclude responses to light stimuli in the electroretinography,amplifying the amplitude of the b-wave.

Recoverin expression was observed in 40 of the total of 168 cells permicroscopic field in the non-transplanted group (positive rate: 23.8%),but in 120 of the total 292 cells per microscopic field in thetransplanted group (positive rate: 41.0%) (p<0.0001) (Table 7 and FIG.21). In the non-transplanted group, recoverin means bipolar cells in theinner nuclear layer and cone photoreceptor cells in the outer nuclearlayer. In the transplanted group, a 4- or 5-fold recoverin-positive celllayer was formed in the outer nuclear layer as well as in the innernuclear layer. From these observations, it is understood that whentransplanted into an injured retina, hESC-derived photoreceptor cellscan graft at proper loci onto the retina and effectively reconstruct theouter nuclear layer which has been lost due to degeneration.

TABLE 7 Evaluation of hESC-Derived Photoreceptor Cells Transplanted intoMice with Retinal Degeneration Non-transplanted Group Transplanted GroupNo. of Total Positive No. of Total Positive Positive Cell Rate PositiveCell Rate Marker Cells Count (%) Cells Count (%) rhodopsin  2 199 1   88215 40.8 recoverin 40 168 23.8 120 292 41.0

In addition, the engrafted cells were observed to express synaptophysin,suggesting that they constructed, in synaptic interaction with otherintraretinal cells, retinal neurons and light circuits (FIG. 22). Thatis, the expression of synaptophysin indicates that the photoreceptorcells in the newly formed outer nuclear layer are using synapses tointeract with other intraretinal cells to function as a circuit inresponse to light.

Taken together, the data obtained above implies that when transplanted,the photoreceptor cells differentiated from hESCs according to thepresent invention can participate in the construction of retinalcircuits, exert their own functions in the transplanted subject, andopen up the new possibility of giving vision to patients suffering fromblindness that is attributed to the loss of photoreceptor cells.

Example 9 Effect of Wnt Signaling Pathway Activator on Proliferation ofRetinal Cells

In order to examine the role of a Wnt signaling pathway indifferentiation from retinal progenitor cells into retinal cells,various combinations of three differentiation factors Wnt3a (W), Shh (S)and retinoic acid (R) were used (FIG. 18 and Table 8). Thedifferentiation factors were added to media according to culture stepand time. Wnt3a was added at a concentration of 50 ng/ml on culture day13, Shh at a concentration of 250 ng/ml on culture day 18, and retinoicacid at a concentration of 500 nM on culture day 21. In each medium,hESCs were cultured for a total of 29 days before cell counting.

When undifferentiated hESCs were seed at a density of 1.3 (±0.1)×10⁶cell per well into 6-well cell culture plates, 303±12 floatingaggregates were formed. For differentiation, 53 of the floatingaggregates (corresponding to 1.55×10⁴ undifferentiated hESCs) wereinoculated onto each well.

Statistical analysis of total cell populations in each medium was doneusing the Kruskal-Wallis test, showing that the five media differed incell population from one to another with statistical significance(p=0.0026). An additional analysis was conducted to examine differencesbetween groups. FDR-adjusted p-values for one error were obtained andare summarized in Table 8, below.

TABLE 8 Effect of Differentiation Factors on Cell Differentiation CellCount (×10⁶)(Mean ± S.E.M.) 3.98 0.73 2.21 2.74 0.87 (±0.64) (±0.16)(±0.67) (±0.36) (±0.38) Main Wnt3a + − + + − Factors (50 ng/ml)Shh + + + − − (250 ng/ml) RA (500 nM) + + − + −

The highest cell population was measured upon the use of W+/S+/R+ (3.98(±0.64)×10⁶ cells), followed by 2.74 (±0.36)×10⁶ cells in W+/S−/R+ and2.21 (±0.67)×10⁶ cells in W+/S+/R−. W−/S−/R− and W−/S+/R+ proliferatedthe cells at a density of as low as 0.87 (±0.38)×10⁶ and 0.73(±0.16)×10⁶, respectively. The data indicate that cell proliferation isindependent of the presence of S and R, but depends on the presence of Wand that a great part of the cell proliferation results from the effectof Wnt3a. When comparing the two media W+/S+/R+ and W−/S+/R+, the cellpopulations (3.98×10⁶ vs. 0.73×10⁶) differ by five times. This isanother evidence that Wnt3a plays a critical role in cell proliferation.

A statistical analysis showed that there was a difference betweenW+/S+/R+ and W−/S+/R+ (p=0.03967), between W+/S+/R+ and W−/S−/R−(p=0.03967), and between W−/S+/R+ and W+/S−/R+ (p=0.03967), withsignificance (p<0.05) (FIG. 21). After being cultured four weeks in the(W+/S+/R+) culture medium for 4 weeks, the undifferentiated cellsproliferated to 3.98×10⁶ differentiated cells, which was 257-fold higherthan the population of the initial cells.

Example 10 Role of Wnt Signaling Pathway Activator in Differentiationinto Retinal Cells

To differentiate the retinal progenitor cells into neural retinalprogenitor cells, Dkk-1 supply was quit on day 13 after induction and aculture medium containing a combination of noggin, IGF-1, FGF2 and Wnt3a(50 ng/ml) was supplied for 5 days. Wnt3a must be removed from theculture media so as to induce and promote the development of theforebrain and eye field precursors during the early embryogenesis.However, Pax6-positive cells increased in a dose-dependent manner withincrease in Wnt3a concentration. Pax6 is expressed upon the developmentof the retina during embryogenesis, that is, on all proliferatingretinal progenitor cells (Marquardt & Gruss. Trends Neurosci., 2002; 25:32-8) in addition to being essential for the generation of neuralretinal progenitor cells. Accordingly, an increased expression level ofPax6 is indicative of highly efficient differentiation into neuralretinal progenitor cells. Wnt3a was supplied, with the concomitantremoval of the Wnt3a inhibitor, in order to induce the expression ofPax6 at a high rate. The expression levels of retinal cell markersbefore and after Wnt3a addition are given in Table 9, below.

TABLE 9 Expression Levels of Retinal Cell Markers before and after Wnt3aAddition Mean ± S.E.M. (%, Sample size = 3) Statistical Before Wnt3 aAfter Wnt3 a addition Significance Marker addition (on Day 13) (on Day18) (p value) Rax 86.6 (±3.0) 98.2 (±0.9) <0.0001 Pax6 63.9 (±0.9) 89.1(±2.5) <0.0001 Otx2 76.4 (±2.0) 61.5 (±0.6) <0.0001 Sox2 83.0 (±1.9)68.1 (±1.1) <0.0001 Chx10 46.3 (±1.0) 64.5 (±1.6) <0.0001 Nestin 65.7(±2.7) 18.0 (±1.4) <0.0001 Mitf 17.2 (±0.4)  2.7 (±1.0) <0.0001 Crx 12.8(±1.6) 80.1 (±0.2) <0.0001 Recoverin 35.8 (±0.4) 68.5 (±2.6) <0.0001Rhodopsin 35.3 (±1.7) 52.9 (±3.4) <0.0001 Peripherin2  5.6 (±0.3) 13.9(±2.4) <0.0001 Ki67 87.5 (±1.3) 31.5 (±0.5) <0.0001

On the differentiated neural retinal progenitor cells, the positiverates were increased simultaneously from 63.9% to 89.1% for Pax 6(p<0.0001), from 86.6% to 98.2% for Rax (p<0.0001), and from 46.3% to64.5% for Chx10 (p<0.0001), indicating that the increase in the positiverate of Pax6 resulted from the proliferation of neural retinalprogenitor cells (both Rax+ and Pax6+ positive) (Table 9).

Nestin, a marker characteristic of most CNS neural progenitoc cells,which is known to be not expressed in neural retinal progenitor cells(Yang et al, Mech. Dev. 2000; 94: 287-91), was greatly decreased inpositive rate from 65.7% to 18.0% after Wnt3a addition (p<0.0001), fromwhich it is understood that the Wnt signaling pathway restrained theproliferation of neural progenitor cells and promoted the proliferationand differentiation of neural retinal cells.

On the other hand, the proliferative cell marker Ki67 rapidly decreasedin positive rate from 87.5% to 31.5% (p<0.0001), indicating thatdifferentiation was more active than proliferation. Immediately afterneural retinal progenitor cells leave the cell proliferation cell cycle,photoreceptor cell precursors are generated with the concomitantexpression of Crx. Thus, the Wnt signaling pathway induced the neuralretinal progenitor cells leaving the cell proliferation cycle todifferentiate into Crx-expressing photoreceptor cell precursors (Table9).

With regard to the signal pathway for inducing differentiation fromretinal progenitor cells into Crx-positive photoreceptor cell precursorsin vivo, its exact biological changes and molecules have not yet beenreported in any prior art, so far (Ikeda et al., Proc. Natl. Acad. Sci.USA, 2005; 102: 11331-6). In the present invention, however, Wnt3a wasfirst found to very strongly induce the expression of the photoreceptorcell precursor marker Crx. That is, the positive rate of Crx showed adrastic increase from 12.8% before Wnt3a addition to 80.1% after Wnt3aaddition (p<0.0001) (Table 9). The increased expression level of Crx hadon influence the differentiation and generation of photoreceptor cells,directly promoting differentiation into photoreceptor cells. Anincreased expression level was also found in the universal photoreceptorcell marker recoverin (from 35.8% to 68.5%, p<0.0001), in the rodphotoreceptor cell marker rhodopsin (from 35.3% to 52.9%, p<0.0001), andin the photoreceptor cell's outer segment marker peripherin2 (from 5.6%to 13.9%, p<0.0001) (Table 9)

Mitf, an antigen marker of retinal pigmented epithelial progenitorcells, showed a decrease in positive rate from 17.2% to 2.7% (p<0.0001),demonstrating that the Wnt signaling pathway promoted the generation ofneural retinal cells and photoreceptor cells, but prevents the formationof retinal pigmented epithelium (Table 9).

Example 11 Role of Inhibitor of Wnt Signaling Pathway Activator inDifferentiation into Retinal Cells

<11-1> Addition of Wnt Signaling Pathway Antagonist

In order to examine the function of the Wnt signaling pathway activatoron differentiation from hESCs into retinal cells, particularlyphotoreceptor cells, cells were cultured in the presence of a Wntsignaling pathway antagonist. The Wnt/Frizzled signaling pathwayinhibitor Dkk-1 (R&D Systems, Minneapolis, Minn., USA) was added at aconcentration of 10 ng/ml, 100 ng/ml or 1 μg/ml on day 13, followed byculturing the cells for an additional 16 days. While the antagonistconcentrations were maintained uniformly over the culture time period,the Wnt signaling pathway activator was assayed for activity. Dkk-1 wasdissolved in 0.1% bovine serum albumin (BSA, Sigma-Aldrich) in PBSbefore use.

According to a protocol for differentiation into retinal cells, Wnt3aand Dkk-1 were added on Day 13 at the following concentrations: 50 ng/mlWnt3a (group I), free of both Wnt3a and Dkk-1 (group II), 10 ng/ml Dkk-1(group III), 100 ng/ml Dkk-1 (group IV), and 1 μ/ml Dkk-1 (group V). Ineach medium, the cells were cultured to Day 29. In this experiment, noneof the other major differentiation factors Shh and RA were used.

<11-2> Immunofluorescent Staning

To examine the effect of the Wnt signaling pathway activator on the celldifferentiation of Example 11-1, an immunofluorescent staining analysiswas performed as follows.

On Day 29, the cells which had been cultured onpoly-D-lysine/laminin-coated, 8-well slides (BD Biosciences) under theconditions of Wnt3a and Dkk-1 (groups I-V) were fixed with 4%paraformaldehyde (Sigma-Aldrich), after which non-specific reactionswere blocked with PBS containing 3% BSA (Jackson ImmunoresearchLaboratory, Bar Harbor, Me., USA) and 0.25% Triton X-100(Sigma-Aldrich).

After being blocked for 90 min, the slides in each differentiation stepwere incubated overnight at 4° C. with the following antibodies specificfor cells of each differentiation step: rabbit-Blue-opsin (1:500,Chemicon), sheep-Chx10 (1:100, Chemicon), rabbit-Crx (1:200, Santa CruzBiotechnology, Santa Cruz, Calif., USA), rabbit-GFAP (1:200,Invitrogen), mouse-human specific mitochondria (1:50, Chemicon),rabbit-human specific mitochondria (1:200, Chemicon), mouse-Islet1(1:10, Developmental Studies Hydroma Bank, DSHB; Iowa City, Iowa, USA),mouse-Ki67 (1:100, Vector Laboratories, Peterborough, England),mouse-Mitf (1:1,000, abcam, Cambridge, Mass., USA), mouse-nestin (1:250,BD Sciences), rabbit-neurofilament-200 (1:1,000, Sigma-Aldrich),rabbit-Otx2 (1:100, abcam), rabbit-Pax2 (1:250, abcam), mouse-Pax6 (1:2,DSHB), mouse-peripherin2 (1:500, GenScript, Piscataway, N.J., USA),rabbit-Rax (1:250, abcam), rabbit-recoverin (1:1,000, Chemicon), retinalpigment epithelium 65 (RPE65, 1:100, Chemicon), rabbit-rhodopsin (1:500,Sigma-Aldrich), mouse-rhodopsin (1:500, Ret-P1, Lab Vision, Fremont,Calif., USA), mouse-rhodopsin (1:2,000, Ret-P1, Sigma-Aldrich),mouse-rom1 (1:50, ABR-Affinity Bioreagents, Golden, Colo., USA),rabbit-PDE6 beta (1:100, abcam), rabbit-phosducin (1:500, Santa CruzBiotechnology), mouse-PKC-alpha (1:500, Sigma-Aldrich), mouse-Prox1(1:2,000, Chemicon), mouse-Sox2 (1:250, R&D Systems),rabbit-synaptophysin (1:2,000, Santa Cruz Biotechnology), andrabbit-ZO-1 (1:100, Zymed-Invitrogen).

Before use, these antibodies were diluted in a PBS solution containing1% BSA and 0.25% Triton X-100. The cells were washed three times for 5min with PBS and incubated at room temperature for 2 hrs withspecies-specific secondary antibody conjugated with Cy3 (1:800, JacksonImmunoresearch Laboratory) or Alexa488 (1:500, Invitrogen). A standardmaterial suitable for the primary and the secondary antibody was used toexamine non-specific staining or interaction between the antibodies.Afterwards, the cells were washed three times for 5 min with PBS,counterstained with DAPI (4,6-diamidino-2 phenylindole) and mounted inVectashield (Vector Laboratories), followed by visualization under anepifluorescence microscope (Nikon Eclipse, E800, Tokyo, Japan) and aconfocal microscope (Leica, Leica Microsystems Inc, Bannockburn, Ill.,USA or Zeiss LSM510, Carl Zeiss, Inc, Thornwood, N.Y., USA).

500 cells were counted from 20 microscopic fields randomly selected at400× magnification and evaluated for positive responses to eachantibody. Positive responses to antibodies were determined after atleast three evaluations. After the data was corrected for clustereffects, statistical analysis of the data was done using the GEE(Generalized Estimating Equations) model of SAS version 9.1 so as toinvestigate changes in positive rate with concentrations of Wnt3a andDkk-1. All data were represented as mean±standard error of the mean(S.E.M) with a statistical significance of p<0.05.

As a result, significant differences were detected among thephotoreceptor cell marker. The photoreceptor cell markers Crx(p=0.0247), recoverin (p=0.0113), rhodopsin (p=0.0166) and peripherin2(p=0.0166) remarkably decreased in expression level with increasing ofDkk-1 concentration. Statistical significances were found between allgroups except for group I vs. group II and group IV vs. group V in Crxand group I vs. group II in recoverin (significance between groups: *:p<0.0001; **: p=0.0381) (Tables 10 and 11 and FIG. 24). These datashowed that the differentiated photoreceptor cells resulted from theactivation of the Wnt signaling pathway and that the inhibition of theWnt signaling pathway led to almost no generation of photoreceptorcells.

The rhodopsin which was expressed at a positive rate of 11.9% in spiteof 1 μg/ml Dkk-1 was thought to be attributed to the stabilization ofbeta-catenin by a Wnt independent mechanism or the activation ofnon-canonical pathway by Wnt, Wnt5 and Wnt11. Also, the expression levelof the retinal progenitor cell marker Pax6 significantly differed fromone group to another over the overall 5 groups (p=0.0275) and wasdecreased with an increase in, Dkk-1 concentration (all p values betweengroups: p<0.0001) (FIG. 24, Tables 9 and 10).

TABLE 10 Positive Rates of Photoreceptor Cell Markers According toConcentrations of Wnt3a and its inhibitor Dkk-1 Mean ± S.E.M. (%, Samplesize = 3) Wnt3a, 50 Wnt3a (−), Dkk-1, 100 ng/ml Dkk-1 (−) Dkk-1, 10ng/ml ng/ml Dkk-1, 1 μg/ml, Marker (Group I) (Group II) (Group III)(Group IV) (Group V) Pax6 82.1 (±4.0) 64.0 (±1.0) 63.9 (±0.9) 51.8(±1.0) 42.1 (±2.1) Crx 32.5 (±0.3) 31.8 (±3.8) 54.3 (±3.1)  7.4 (±1.1) 7.5 (±0.9) Recoverin 72.3 (±0.8) 71.1 (±0.8) 55.1 (±0.6) 41.5 (±1.3)15.2 (±4.0) Rhodopsin 73.8 (±0.9) 52.7 (±1.0) 49.1 (±2.0) 28.9 (±0.5)11.9 (±3.0) Peripherin2 21.5 (±0.3) 16.4 (±0.8) 27.0 (±1.0)  2.1 (±1.0) 0.1 (±0.1)

TABLE 11 Statistical Significances of Positive Rates of PhotoreceptorCell Markers According to Concentrations of Wnt3a and its InhibitorDkk-1 Statistical Significance (p value) Group I vs. Group II vs. GroupIII vs. Group IV vs. Marker Group II Group III Group IV Group V Pax6<0.0001 0.9036 <0.0001 <0.0001 Crx 0.8147 <0.0001 <0.0001 1.0000Recoverin 0.1864 <0.0001 <0.0001 <0.0001 Rhodopsin <0.0001 0.0381<0.0001 <0.0001 Peripherin2 <0.0001 <0.0001 <0.0001 <0.0001 *Statisticalsignificance: p < 0.05 *Group I: Wnt3a, 50 ng/ml; Group II: Wnt3a (−),Dkk-1 (−); Group III: Dkk-1, 10 ng/ml; Group IV: Dkk-1, 100 ng/ml; GroupV: Dkk-1, 1 μg/ml.

Example 12 Positive Rates of Retinal Cell Markers Upon Use ofSubstitutes for Wnt3a and Shh

Differentiation from retinal progenitor cells into neural retinalprogenitor cells, from neural retinal progenitor cells intophotoreceptor cell precursors, and from photoreceptor cells intophotoreceptor cells was induced in the same manner as in Examples 4 to6, with the exception that the media used in Examples 4 to 6 contained50 ng/ml recombinant Wnt1 (PeproTech, Rocky Hill, N.J., USA), 50 or 100ng/ml recombinant Wnt5A (R&D Systems), 50 or 100 ng/ml recombinant Wnt11(R&D Systems), 50 ng/ml recombinant Norrin (R&D Systems), 2.5 mM and 5mM LiCl (Sigma-Aldrich), 2 μM BIO (Sigma-Aldrich), or 30 μM SB415286(Sigma-Aldrich), instead of the recombinant Wnt3a, and with the furtherexception that the media used in Examples 5 and 6 contained 1 μMpurmorphamine (Stemgent, Cambridge, Mass., USA) instead of therecombinant Shh.

The cells differentiated with various substitutes for Wnt3a and Shh weresubjected to immunochemical staining in the same manner as in Example7-1 and assayed for positive rates of retinal cell markers, and theresults are summarized in Tables 12 and 13, below.

TABLE 12 Positive Rates of Retinal Cell Markers upon Use of Wnt3aSubstitutes Mean ± S.E.M (%, Sample size = 3) Substitute recoverinrhodopsin Rom peripherin2 Crx Ki67 Pax6 b-opsin Wnt1 70.9(±1.8)66.2(±5.4) 55.9(±2.9)  3.8(±2.0) 60.3(±1.3) 23.8(±1.2) ND 61.6(±6.8) (50ng/ml) Wnt5A 61.0(±3.2) 59.5(±2.0) 58.8(±2.7) 20.7(±0.8) 67.3(±1.9)41.3(±3.9) ND 55.0(±4.4) (50 ng/mL) Wnt5A 61.6(±2.5) 56.1(±1.8) ND14.2(±2.8) ND ND ND ND (100 ng/ml) Wnt11 65.3(±2.7) 51.8(±7.7)50.2(±7.4)  0.5(±0.3) 65.9(±5.8) 40.1(±5.2) ND 30.6(±6.2) (50 ng/ml)Wnt11 61.8(±4.1) 59.1(±5.4) ND 10.7(±2.9) ND ND ND ND (100 ng/ml) Norrin85.0(±7.2) 82.9(±7.1) 69.5(±2.7) 24.9(±4.5) 39.3(±0.8) 42.2(±6.8) ND71.1(±0.6) (50 ng/ml) BIO (2 uM) 77.1(±0.8) 71.5(±1.2) 60.3(±0.9)33.9(±0.1) 65.8(±1.0) 17.2(±0.6) 92.8(±1.0) 77.5(±1.8) SB41528670.0(±0.6) 69.3(±0.5) 59.5(±0.7)  9.1(±0.6) 53.7(±2.7) 13.3(±1.2)89.1(±0.6) 53.7(±2.7) (30 uM) LiCl 64.1(±2.9) 56.2(±1.7) ND ND ND ND NDND (2.5 mM) LiCl 78.9(±4.4) 68.5(±2.2) ND  1.5(±0.9)  5.0(±1.7)33.3(±2.2) ND ND (5 mM) *ND: Non-determined.

TABLE 13 Positive Rates of Retinal Cell Markers upon Use of ShhSubstitutes Mean ± S.E.M (%, Sample size = 3) Substitute recoverinrhodopsin peripherin2 Crx Ki67 purmorpha- 76.5(±0.9) 73.5(±1.2)41.5(±1.2) 71.6(±0.8) 24.3(±0.5) mine (1 uM)

As is apparent from the data of Tables 12 and 13, the Wnt3a substitutes,Wnt1, Wnt5a, Wnt11, norrin, LiCl, BIO and SB415286 and the Shhsubstitute purmorphamine allowed the retinal cell markers to beexpressed at similar positive rates to those when Wnt3a and Shh wereused.

The invention claimed is:
 1. A method for producing retinal cellscomprising: a) culturing the human ESCs or IPSCs as floating aggregatesin a medium comprising IGF-1 DKK-1, and NOGGIN to differentiate thehuman embryonic stem cells into eye field precursor cells; b) culturingthe eye field precursor cell floating aggregates in a medium comprisingIGF-1 DKK-1, NOGGIN, and FGF-2 to differentiate the eye field precursorcells into retinal progenitor cells; c) inducing differentiation of theretinal progenitor cells to retinal cells by culturing the retinalprogenitor cells in a medium comprising a differentiation factorselected from the group consisting of: a Wnt signaling pathwayactivator; wherein the medium does not comprise exogenous retinoic acidand exogenous sonic hedgehog.
 2. The method as set forth in claim 1,wherein the retinal cells are photoreceptor cells.
 3. The method as setforth in claim 1, wherein the Wnt signaling pathway activator isselected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a,Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b,Wnt10a, Wnt10b, Wnt11, Wnt16b; β-catenin expression level-increasingsubstances; Axin inhibitors; APC (adenomatous polyposis coli)inhibitors; norrin; R-spondin 2; and a combination thereof.
 4. Themethod as set forth in claim 3, the Wnt signaling pathway activator isselected from the group consisting of Wnt1, Wnt3a, Wnt5a, Wnt11, norrinand a combination thereof.
 5. The method as set forth in claim 1,wherein the Wnt signaling pathway activator is a GSK3 (glycogen synthasekinase 3) inhibitor.
 6. The method as set forth in claim 5, wherein theGSK3 inhibitor is selected from the group consisting of lithium (Li),LiCl, bivalent zinc (bivalent Zn), BIO (6-bromoindirubin-3′-oxime),SB216763, SB415286, CHIR99021, QS11 hydrate, TWS119, kenpaullone,alsterpaullone, indirubin-3′-oxime, TDZD-8, Ro 31-8220 methanesulfonatesalt and a combination thereof.
 7. The method as set forth in claim 6,wherein the GSK3 inhibitor is selected from the group consisting ofLiCl, BIO (6-bromoindirubin-3′-oxime), SB415286 and a combinationthereof.
 8. The method as set forth in claim 1, wherein theconcentration of the Wnt signaling pathway activator used in the mediumis ranging from 0.01 to 500 ng/ml.
 9. The method as set forth in claim8, the concentration of the Wnt signaling pathway activator used in themedium is ranging from 1 to 100 ng/ml.
 10. The method as set forth inclaim 6, wherein the concentration of the GSK inhibitor except for LiCl,BIO and SB415286 used in the medium is ranging from 0.01 to 500 ng/ml.11. The method as set forth in claim 10, wherein the concentration ofthe GSK inhibitor except for LiCl, BIO and SB415286 used in the mediumis ranging from 1 to 100 ng/ml.
 12. The method as set forth in claim 6,wherein the concentration of LiCl used in the medium is ranging from 0.1to 50 mM; that of BIO is ranging from 0.1 to 50 μM; and that of SB415286is ranging from 0.1 to 500 μM.
 13. The method as set forth in claim 12,wherein the concentration of LiCl used in the medium is ranging from 1to 10 mM; that of BIO is ranging from 0.5 to 5 μM; and that of SB415286is ranging from 5 to 50 μM.